Modulators of myocyte lipid accumulation and insulin resistance and methods of use thereof

ABSTRACT

Formulations and methods for reducing blood glucose and/or increasing insulin signaling in a subject have been developed. The formulations include SBI-477 and compounds based on SBI-477 i.e., SBI-477 analogs (collectively, SBI-477 compounds) and/or Mondo family inhibitors, in an effective amount to inhibit intracellular lipid accumulation and/or increase cellular glucose uptake when compared to levels in a control subject not administered the composition. Also disclosed are methods of reducing intracellular lipid accumulation and/or increase glucose uptake in a subject in need thereof. The method includes administering to the subject an effective amount of SBI-477 compounds and/or Mondo family inhibitor to reducing intracellular lipid accumulation and/or increase glucose uptake in the subject. Also disclosed are method for treating one or more Myc-driven cancers, including neuroblastoma, lung squamous cell carcinoma/lung adenocarcinoma, liver hepatocellular carcinoma, colon adenocarcinoma, acute myeloid leukemia, and breast invasive carcinoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/197,534, filed Jul. 27, 2015. Application No. 62/197,534, filed Jul.27, 2015, is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos. R01DK045416, R24 DK092781, and R24 DK084969 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on Jul. 27, 2016 as a text file named“SBMRI_14-022_PCT_ST25,” created on Jul. 26, 2016, and having a size of13,695 bytes is hereby incorporated by reference.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of compositions andmethods of reducing one or more symptoms associated with insulinresistance.

BACKGROUND OF THE INVENTION

The rising prevalence of obesity is driving an alarming increase in type2 diabetes, a global health threat. Co-morbidities associated withobesity include insulin resistance, fatty liver disease (NAFLD/NASH) andlipotoxic cardiomyopathy. The development of obesity-related diabetesrepresents a final common pathogenic pathway that further contributes tothe comorbid diseases. Accordingly, delineation of the mechanismsinvolved in the development of insulin resistance is a critical steptowards the identification of new therapeutic targets aimed at the earlytreatment of this progressive feed-forward disease process.

The development of insulin resistance is strongly associated withaccumulation of intracellular lipid in tissues outside of adiposeincluding skeletal muscle, liver, and heart. In obese humans,intramyocellular lipid (IMCL) is negatively correlated with whole-bodyinsulin sensitivity (Pan, et al., Diabetes. 1997; 46(6):983-988; Krssak,et al., Diabetologia. 1999; 42(1):113-116 and Goodpaster et al.,Metabolism. 2000; 49(4):467-472). The skeletal myocyte imports fattyacids (FA) into the cell from circulating free fatty acid (FAs) orlipoprotein particles, such as very low density lipoprotein (VLDL), tosupport energy production. Once transported into the cell, FAs areoxidized for ATP production, used to build membranes, or stored astriglyceride. However, excessive skeletal myocyte lipid delivery, suchas occurs in the obese state, leads to expansion of IMCL. Increasedimport of fatty acids is thought to initially trigger an adaptiveresponse within the skeletal muscle to increase capacity formitochondrial fatty acid oxidation (Mitra, et al. Mol Cell Cardiol.2012; 52(3):701-710). In the long-term, however, increased delivery offatty acids can exceed mitochondrial oxidative capacity setting thestage for a “vicious cycle” of cellular lipotoxicity, leading to insulinresistance. In support of this notion, some studies have shown thatmitochondrial oxidative capacity is reduced in insulin resistantdiabetic subjects (Petersen, et al., New Engl J Med. 2004;350(7):664-671; Schrauwen-Hinderling, et al., Diabetologia. 2007;50(1):113-120 and Phielix, et al., Diabetologia. 2007; 50(1):113-120).

The mechanistic links between IMCL and the development of insulinresistance are poorly understood. The results of studies to date suggestthat the lipid storage depot per se is likely not a culprit in thegenesis of cellular “lipotoxicity” and insulin resistance. Indeed, somestudies have suggested that capacity to store lipid within the cellserves a protective function (Listenberger, et al., Proc Natl Acad SciUSA. 2003; 100(6):3077-3082; Liu, et al., J Biol Chem. 2009;284(52):36312-36323). Rather, generation and accumulation of lipidintermediates have been proposed to alter insulin stimulated glucoseuptake (Samuel, et al., Biol Chem. 2004; 279(31):32345-32353; and Bosma,et al. Prog Lipid Res. 2012; 51(1):36-49). For example, lipid-deriveddiacylglycerol (DAG) species have been shown to activate protein kinaseC-ε and θ isoforms to phosphorylate the insulin receptor substrate-1(IRS-1), blocking the actions of the insulin receptor (Idris, et al.,Ann N Y Acad Sci. 2002; 967:176-182; Kumashiro et al., Proc Natl AcadSci USA. 2011; 108(39):16381-16385). Ceramides and reactive oxygenspecies have also been shown to inhibit insulin signaling in certaincontexts (Chavez, et al., J Biol Chem. 2003; 278(12):10297-10303;Anderson, et al., J Clin Invest. 2009; 119(3):573-581). In addition,intermediates of incomplete fatty acid oxidation have been implicated ininsulin resistance (Koves, et al., Cell Metab. 2008; 7(1):45-56).However, the role of such processes as primary drivers of insulinresistance related to altered cellular lipid balance (i.e., causes)versus serving as downstream effectors (i.e., effects) has been unclear.Moreover, regulatory circuitry that links control of cellular lipidbalance and insulin signaling—which would enable identification of moreeffective therapeutic intervention methods—has not been identified.There is a still a need for identification of compounds that not onlyinhibit intramyocellular lipid accumulation but also increase glucoseuptake.

It is an object of the present invention to provide compounds whichinhibit intracellular lipid.

It is also an object of the present invention to provide compounds whichreduce intracellular lipid and increase cellular glucose uptake andinsulin signaling.

It is also an object of the present invention to provide small moleculeinhibitors of the Mondo transcription factors.

It is also an object of the present invention to provide a method ofreducing intracellular lipid in a subject in need thereof.

It is also an object of the present invention to provide a method ofincreasing insulin sensitivity in a subject in need thereof.

BRIEF SUMMARY OF THE INVENTION

Formulations and methods for reducing blood glucose, increasing insulinsignaling, or combinations thereof, in a subject have been developed.The formulations include SBI-477 and compounds based on SBI-477, i.e.,SBI-477 analogs (collectively, SBI-477 compounds), Mondo transcriptionfactor inhibitors, or combinations thereof, in an effective amount toinhibit intracellular lipid accumulation, increase cellular glucoseuptake, or combinations thereof, when compared to levels in a controlsubject not administered the composition. In some forms, the compoundsare present in an effective amount to inhibit TAG synthesis. In someforms, the compounds are present in an effective amount to increasecellular glucose uptake, for example, by enhancing insulin signaling.Preferably, the formulation is effective to inhibit cellular TAGsynthesis and increase cellular glucose uptake. In some forms, thecompounds are present in an effective amount to inhibit nuclearlocalization of MondoA and/or MondoB. In preferred forms, the compoundsreduce intramyocellular lipid, measured as, for example, TAG levels.Preferred compounds are SBI-477 and SBI-993, the structures of which aredisclosed below.

Also disclosed are methods of reducing intracellular lipid accumulation,increase glucose uptake, or combinations thereof, in a subject in needthereof. The method includes administering to the subject an effectiveamount of SBI-477 compounds, a Mondo transcription factor inhibitor, orcombinations thereof, to reducing intracellular lipid accumulation,increase glucose uptake, or combinations thereof, in the subject. Inpreferred forms, the subject is obese, has type-2 diabetes orpre-diabetes, or suffers from a condition associated with lipidtoxicity.

Also disclosed are methods for treating one or more Myc-driven cancers,including neuroblastoma, lung squamous cell carcinoma/lungadenocarcinoma, liver hepatocellular carcinoma, colon adenocarcinoma,acute myeloid leukemia, and breast invasive carcinoma. The methodincludes administering the compositions and formulations disclosedherein to a subject in need thereof, where the formulations include thedisclosed compounds in an effective amount to inhibit MondoA and/orMondoB.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several forms and embodiments ofthe disclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIGS. 1A-1C show SBI-477 as a small molecule inhibitor of neutral lipidaccumulation in human skeletal myotubes. FIG. 1A shows the effects ofSBI-477, over a dose range, on triglyceride levels in human skeletalmyotubes following 24 hour exposure to 100 μM oleate. Data is shown aspercent of DMSO vehicle control. Representative of >5 experiments. FIGS.1B and 1C show dose-dependent inhibition of myocyte TAG accumulation bySBI-477. FIG. 1B—primary human myotubes were incubated with theindicated concentrations of SBI-477, 1 μM triascin C, 1 μM A922500 (DGATinhibitor) or vehicle control with 100 μM oleate for 24 hours. Fattyacid free bovine serum albumin (BSA) was included as non-lipid loadingcontrol. Bars represent mean triglyceride level normalized to totalprotein±SD. *p<0.001 vs. oleate/vehicle control by one-way ANOVA withBonferroni post hoc test. FIG. 1C—primary human myotubes were incubatedwith 10 μM SBI-477 or vehicle control for 24 hours. Fatty acid (FA)uptake was measured as cellular uptake of 3H-oleate. Bars representfatty acid uptake normalized to the vehicle control. FIGS. 1D-1F showthe effect of SBI-477 on FAO rates in human skeletal myotubes. FIG.1D—human skeletal myotubes were incubated with the indicatedconcentration of SBI-477, AICAR (1 mM) or etomoxir (50 μM) for 24 hours.FAO rates were determined by 3H-palmitate oxidation (n=5). AICAR andetomoxir were included as controls for activation and inhibition of FAO,respectively. *p<0.01 vs. vehicle. Total cellular triglyceride levelswere determined following incubation with SBI-477 (10 μM) for 24 hourswith etomoxir (50 μM) in the absence (FIG. 1E) or presence of 1 mMcarnitine (FIG. 1F) (n=4). *p<0.05 vs. vehicle control.

FIGS. 2A and 2B show SBI-477 inhibition of expansion of cellular DAG andTAG pools in oleate-loaded myocytes. The results of quantitativelipidomic analyses performed on human skeletal myotubes exposed tobovine albumin serum (BSA) or 100 μM oleate in the presence of DMSOvehicle (white bars) or 10 μM SBI-477 (black bars) for 24 hours. (FIG.2A) Total mean TAG and DAG levels are shown. (FIG. 2B) Levels ofindividual TAG fatty acyl species with 18:1 species shown separately(left). The data represents mean±SD (n=3). *p<0.05 vs. vehicle, †p<0.05vs. vehicle oleate loaded by Student's t-test.

FIGS. 3A, B, C, E and F show SBI-477 stimulation of glucose uptake andactivates insulin signaling in the absence of insulin. Human skeletalmyotubes were incubated with SBI-477 at the indicated concentration for24 hours and then treated with or without insulin (100 nM) for 30minutes. Glucose ([3H]-2-deoxyglucose, 2-DG) uptake (FIG. 3A) andglycogen synthesis rates (FIGS. 3B and 3C) were measured (n=5) asdescribed in Methods. *p<0.05 vs. Vehicle, no insulin, †p<0.05 vs.Vehicle/insulin by two-way ANOVA with Tukey's post hoc test. (FIG. 3E)Western blot analysis of human myotubes treated with SBI-477 for 24hours was performed to determine the effect on steps of the insulinsignaling pathway using specific Akt and IRS-1 phosphorylation sites asendpoints. Insulin treatment (100 nM) for 30 minutes was used aspositive control. (FIG. 3F) Quantitation of the western blot data in(FIG. 3D) is shown (n=5). *p<0.05 or **p<0.01 vs. Vehicle by one-wayANOVA with Bonferroni post hoc test. The data represent mean±SD.

FIGS. 3D and G show SBI-477-mediated increase in glucose uptake inoleate-loaded skeletal myotubes (FIG. 3D) SBI-477 increases glucoseuptake in oleate-loaded human skeletal myotubes. Basal andinsulin-stimulated [3H]2-DG uptake was measured in human skeletalmyotubes following treatment with SBI-477 for 24 hours in the presenceof 100 μM oleate or BSA as a control. *p<0.05 vs. vehicle, †p<0.05 vs.vehicle/insulin by one-way ANOVA with Bonferroni post hoc test. FIG.3G—Western blot analysis (left) of human myotubes treated with SBI-477for 24 hours was performed to determine activation of S6K and AMPK usingthe indicated phospho-specific antibodies. Insulin treatment (100 nM)for 30 minutes was used as positive control. Quantitation (right) of theWestern blot data is shown (n=4-5). *p<0.05 vs. Vehicle by one-way ANOVAwith Bonferroni post hoc test. The data represent mean±SD.

FIGS. 4A-C show downregulation of TXNIP and ARRDC4 expression bySBI-477. (FIG. 4A) TXNIP and ARRDC4 mRNA levels as determined byquantitative RT-PCR in human myotubes treated with SBI-477 (10 μM) orDGAT1 inhibitor (DGATi, 1 mM) for 24 hours in the absence or presence of100 μM oleate (n=4). Expression is shown relative to vehicle/BSAtreatment. (FIG. 4B) TXNIP gene expression following exposure to a doserange of SBI-477 for 24 hours (n=4). (FIG. 4C) Left, Effect of SBI-477on TXNIP protein levels as determined by western blot analysis bySBI-477. Right, Quantitation of the TXNIP western blot data is shown(n=5). *p<0.05 vs. Vehicle/BSA control, †p<0.05 vs. Vehicle/oleate byone-way ANOVA with Bonferroni post hoc test. The data are shown asmean±SD.

FIGS. 5A-5D show SBI-477 inhibition of MondoA-mediated activation of theTXNIP gene promoter via effects on nuclear localization. FIG. 5A—Aluciferase reporter construct containing approximately 1.5 kb of thehuman TXNIP promoter (shown schematically at top) or a pGL3 controlvector was transfected into H9c2 skeletal myocytes. The activity of theTXNIP promoter (relative luciferase units, RLU) was measured followingtreatment with SBI-477 at the indicated concentration (n=5). FIG. 5B—Theactivity of wild-type versus ChoRE mutant TXNIP promoters was measuredin the presence and absence of SBI-477 (10 μM) for 24 hours (n=5). Thered “X” notes inactivity mutations. FIG. 5C ChIP-qPCR analysis wasperformed with anti-MondoA (black bars) or IgG (open bars) controlantibodies in human skeletal myotubes. Occupancy of MondoA on the ChoREof the TXNIP or ARRDC4 promoter following treatment with SBI-477treatment in the absence or presence of oleate is shown (n=4).Occupation on a Mef2 binding site within the IMPA2 promoter is shown asa negative control. FIG. 5D—Western blot analysis was performed on totalcell lysate, and nuclear or cytoplasmic fractions from primary humanmyotubes treated with 10 μM SBI-477 or DMSO vehicle control for 24hours. Lamin A/C and GAPDH were included as controls for the nuclear andcytoplasmic fractions, respectively. *p<0.05 vs. Vehicle by one-wayANOVA with Bonferroni post hoc test. The data represent mean±SD. HSE,heat shock element; ChoRE, carbohydrate response element; PPAR,peroxisome proliferator-activated receptor, JMPA2, inositol(myo)-1 (or4)-monophosphate 2.

FIGS. 6A to 6F show that MondoA depletion mimics action of SBI-477 inhuman skeletal myotubes. TXNIP and ARRDC4 gene expression was measuredfollowing treatment with 10 mM SBI-477 (FIG. 6A) or siRNA-mediatedMondoA (MLXJP) KD (FIG. 6B) (n=4). *p<0.05 vs. Vehicle or non-targetingsiRNA control (siCon). (FIG. 6C) 2-DG uptake following MondoA KD (orsiCon) in the absence or presence of insulin (n=5). *p<0.05 vs.siCon/Vehicle, †p<0.05 vs. siCon/insulin. (FIG. 6D) Cellulartriglyceride levels following MondoA KD in the absence or presence of100 μM oleate (n=5). *p<0.05 vs. siCon/Oleate. Effect of MondoA KD (FIG.6E) or SBI-477 treatment (FIG. 6F) on the expression of genes encodinglipogenic and triglyceride synthesis enzymes (n=4) is shown. *p<0.05 vs.Veh or siCon. The data represents mean±SD. All statistical significancedetermined by Mann Whitney test. FIG. 6G shows MondoA and TXNIP proteinlevels as determined by immunoblotting on day 3, 4, and 5 after siMondoAor non-targeting control siRNA (siCon) knockdown in human myotubes. FIG.6H shows a dose-response curve for the inhibition of triglycerideaccumulation for SBI-993 in human skeletal myotubes.

FIG. 6I shows body weight of mice over the study beginning at 7 weeks ofage and start of high-fat diet (HFD) (n=6-10). Data represents mean±SEM.*p<0.05 vs. CD-vehicle; †p<0.05 vs. HFD-vehicle by one-way ANOVA.

FIGS. 7A-D show SBI-993 inhibition of muscle and hepatic TAGaccumulation and reduces MondoA target gene expression in vivo. Micemaintained on a high-fat diet for 8 weeks were administered SBI-993 (50mg/kg, q.d., s.c.) or a vehicle (Veh) control for the final week ofhigh-fat feeding. Target gene expression (FIG. 7A) and totaltriglyceride content (FIG. 7B) were measured in skeletal muscle(gastroenemius) and liver in mice maintained on control diet (CD) orhigh-fat diet (HFD) (n=6-10 mice/group). *p<0.05 vs. CD/Veh, †p<0.05 vs.HFD/Veh by one-way ANOVA with Bonferroni post hoc test. FIG. 7C—Bloodglucose levels are shown following a glucose tolerance test (1 g/kgglucose, i.p.) after dosing with SBI-993 or vehicle (n=6/group). Datarepresents mean±SEM. *p<0.05 HFD/Veh vs. HFD/SBI-993 by a two-way ANOVAwith Tukey multiple comparison post hoc test. FIG. 7D—Western blotanalysis of gastrocnemius skeletal muscle whole cell lysate from micereceiving an acute insulin challenge (1.5 U/kg for 10 minutes) toexamine insulin signaling using phosphorylated Akt (5473). Top panel isquantification of western blot analysis (n=4-6/condition).Representative western blots are shown in the bottom panel. *p<0.05 vs.CD/vehicle and †p<0.05 vs. HFD/vehicle by one-way ANOVA with Bonferronipost hoc test. CD, control diet; HFD, high-fat diet. FIG. 7E showsSBI-993 reduction of ChREBP and MondoA occupation on target genepromoter elements in liver. ChIP-qPCR analysis was performed withnuclear extracts from liver tissue of mice following 1 weekadministration of SBI-993 or a vehicle control. Antibodies directedagainst ChREBP (black bars), MondoA (gray bars) or an IgG(open bars)control were used. Occupation of carbohydrate response elements from theTxnip and pyruvate kinase (Pklr) gene promoters is shown as % input.Occupation of an unrelated region within intron 26 of the Myh7 gene wasused a negative control. *p<0.05 vs. Veh-HFD for indicated antibody byone-way ANOVA followed by Bonferroni post hoc test.CD, control diet;HFD, high fat diet. FIG. 7F shows SBI-993 inhibition of hepaticsteatosis. Western blot analysis of liver whole cell lysates from micereceiving an acute insulin challenge (1.5 U/kg for 10 minutes) toexamine insulin signaling using phosphorylated Akt (5473). Top panel isquantification of western blot analysis (n=4-6/condition).Representative western blots are shown in the bottom panel.

FIG. 8 is a schematic showing how MondoA directs myocyte fuelhomeostatic checkpoint functions. The proposed gene regulatory (redarrows) and metabolic “checkpoint” responses (blue arrows) downstream ofMondoA are shown. MondoA is a glucose “sensor”, directly activated byglycolytic metabolites that stimulate nuclear import of MondoA. Onceactivated, MondoA functions as a “brake” to limit carbon entry into thecell via increasing levels of TXNIP, an inhibitor of insulin signalingand glucose uptake. In addition, MondoA promotes energy storage throughactivation of enzymes involved in lipid and glycogen synthesis. Thus,MondoA may serve to limit carbon intake and fuel burning duringconditions of “plenty”. However, in states of chronic nutrient excess,persistent activation of MondoA may become maladaptive, contributing toa vicious cycle of cellular lipid accumulation (TAG synthesis) andinsulin resistance (TXNIP-mediated suppressive effects). FFA, free fattyacid; VLDL, very low-density lipoprotein; ChoRE, carbohydrate responseelement; TXNIP, thioredoxin-interacting protein.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particular forms andembodiments and the Example included therein and to the Figures andtheir previous and following description.

I. Definitions

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular forms and embodimentsonly and is not intended to be limiting.

“Substituted,” as used herein, refers to all permissible substituents ofthe compounds or functional groups described herein. In the broadestsense, the permissible substituents include acyclic and cyclic, branchedand unbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, preferably 1-14 carbonatoms, and optionally include one or more heteroatoms such as oxygen,sulfur, or nitrogen grouping in linear, branched, or cyclic structuralformats. Representative substituents include alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl,substituted phenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy,substituted phenoxy, aroxy, substituted aroxy, alkylthio, substitutedalkylthio, phenylthio, substituted phenylthio, arylthio, substitutedarylthio, cyano, isocyano, substituted isocyano, carbonyl, substitutedcarbonyl, carboxyl, substituted carboxyl, amino, substituted amino,amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid,poly(lactic-co-glycolic acid), peptide, and polypeptide groups. Suchalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substitutedalkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy,alkylthio, substituted alkylthio, phenylthio, substituted phenylthio,arylthio, substituted arylthio, cyano, isocyano, substituted isocyano,carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino,substituted amino, amido, substituted amido, sulfonyl, substitutedsulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl,substituted phosphonyl, polyaryl, substituted polyaryl, C₃-C₂₀ cyclic,substituted C₃-C₂₀ cyclic, heterocyclic, substituted heterocyclic, aminoacid, poly(lactic-co-glycolic acid), peptide, and polypeptide groups canbe further substituted. Heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. It isunderstood that “substitution” or “substituted” includes the implicitproviso that such substitution is in accordance with permitted valenceof the substituted atom and the substituent, and that the substitutionresults in a stable compound, i.e. a compound that does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

“Alkyl,” as used herein, refers to the radical of saturated aliphaticgroups, including straight-chain alkyl, alkenyl, or alkynyl groups,branched-chain alkyl, cycloalkyl (alicyclic), alkyl substitutedcycloalkyl groups, and cycloalkyl substituted alkyl. In preferred forms,a straight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), preferably 20 or fewer, more preferably 15 or fewer, mostpreferably 10 or fewer. Alkyl includes methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.

Likewise, preferred cycloalkyls have from 3-10 carbon atoms in theirring structure, and more preferably have 5, 6 or 7 carbons in the ringstructure. The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls,” the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino,amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,aralkyl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred forms, a substituent designatedherein as alkyl is a lower alkyl.

“Alkyl” includes one or more substitutions at one or more carbon atomsof the hydrocarbon radical as well as heteroalkyls. Suitablesubstituents include, but are not limited to, halogens, such asfluorine, chlorine, bromine, or iodine; hydroxyl; —NRR′, wherein R andR′ are independently hydrogen, alkyl, or aryl, and wherein the nitrogenatom is optionally quaternized; —SR, wherein R is hydrogen, alkyl, oraryl; —CN; —NO₂; —COOH; carboxylate; —COR, —COOR, or —CON(R)₂, wherein Ris hydrogen, alkyl, or aryl; azide, aralkyl, alkoxyl, imino,phosphonate, phosphinate, silyl, ether, sulfonyl, sulfonamido,heterocyclyl, aromatic or heteroaromatic moieties, haloalkyl (such as—CF₃, —CH₂—CF₃, —CCl₃); —CN; —NCOCOCH₂CH₂; —NCOCOCHCH; —NCS; andcombinations thereof.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), haloalkyls, —CN and the like. Cycloalkyls canbe substituted in the same manner.

“Heteroalkyl,” as used herein, refers to straight or branched chain, orcyclic carbon-containing radicals, or combinations thereof, containingat least one heteroatom. Suitable heteroatoms include, but are notlimited to, O, N, Si, P and S, wherein the nitrogen, phosphorous andsulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized.

The terms “alkoxyl” or “alkoxy,” “aroxy” or “aryloxy,” generallydescribe compounds represented by the formula —OR, wherein R includes,but is not limited to, substituted or unsubstituted alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, cycloalkenyl, heterocycloalkenyl,aryl, heteroaryl, arylalkyl, heteroalkyls, alkylaryl, alkylheteroaryl.

The terms “alkoxyl” or “alkoxy” as used herein refer to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, and —O-alkynyl. The termalkoxy also includes cycloalkyl, heterocyclyl, cycloalkenyl,heterocycloalkenyl, and arylalkyl having an oxygen radical attached toat least one of the carbon atoms, as valency permits. A “lower alkoxy”group is an alkoxy group containing from one to six carbon atoms.

The term “substituted alkoxy” refers to an alkoxy group having one ormore substituents replacing one or more hydrogen atoms on one or morecarbons of the alkoxy backbone. Such substituents include, but are notlimited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms and structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (AB)C=C(CD) areintended to include both the E and Z isomers. This may be presumed instructural formulae herein wherein an asymmetric alkene is present, orit may be explicitly indicated by the bond symbol C.

The term “alkynyl group” as used herein is a hydrocarbon group of 2 to24 carbon atoms and a structural formula containing at least onecarbon-carbon triple bond.

The term “aryl” as used herein is any C₅-C₂₆ carbon-based aromaticgroup, fused aromatic, fused heterocyclic, or biaromatic ring systems.Broadly defined, “aryl,” as used herein, includes 5-, 6-, 7-, 8-, 9-,10-, 14-, 18-, and 24-membered single-ring aromatic groups, including,but not limited to, benzene, naphthalene, anthracene, phenanthrene,chrysene, pyrene, corannulene, coronene, etc. “Aryl” further encompassespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings (i.e., “fused rings”)wherein at least one of the rings is aromatic, e.g., the other cyclicring or rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocycles. The aryl group can be substituted with one or moregroups including, but not limited to, alkyl, alkynyl, alkenyl, aryl,halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid,or alkoxy.

The term “substituted aryl” refers to an aryl group, wherein one or morehydrogen atoms on one or more aromatic rings are substituted with one ormore substituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, carbonyl (suchas a ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl, or an acyl),silyl, ether, ester, thiocarbonyl (such as a thioester, a thioacetate,or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate,phosphinate, amino (or quarternized amino), amido, amidine, imine,cyano, nitro, azido, sulfhydryl, imino, alkylthio, sulfate, sulfonate,sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkylaryl, haloalkyl(such as CF₃, —CH₂—CF₃, —CCl₃), —CN, aryl, heteroaryl, and combinationsthereof.

“Heterocycle,” “heterocyclic” and “heterocyclyl” are usedinterchangeably, and refer to a cyclic radical attached via a ringcarbon or nitrogen atom of a monocyclic or bicyclic ring containing 3-10ring atoms, and preferably from 5-6 ring atoms, consisting of carbon andone to four heteroatoms each selected from the group consisting ofnon-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O,C₁-C₁₀ alkyl, phenyl or benzyl, and optionally containing 1-3 doublebonds and optionally substituted with one or more substituents.Heterocyclyl are distinguished from heteroaryl by definition. Examplesof heterocycles include, but are not limited to piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl,dihydrofuro[2,3-b]tetrahydrofuran, morpholinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pyranyl,2H-pyrrolyl, 4H-quinolizinyl, quinuclidinyl, tetrahydrofuranyl,6H-1,2,5-thiadiazinyl. Heterocyclic groups can optionally be substitutedwith one or more substituents as defined above for alkyl and aryl.

The term “heteroaryl” refers to C₅-C₂₆-membered aromatic, fusedaromatic, biaromatic ring systems, or combinations thereof, in which oneor more carbon atoms on one or more aromatic ring structures have beensubstituted with a heteroatom. Suitable heteroatoms include, but are notlimited to, oxygen, sulfur, and nitrogen. Broadly defined, “heteroaryl,”as used herein, includes 5-, 6-, 7-, 8-, 9-, 10-, 14-, 18-, and24-membered single-ring aromatic groups that may include from one tofour heteroatoms, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine,pyridazine and pyrimidine, and the like. The heteroaryl group may alsobe referred to as “aryl heterocycles” or “heteroaromatics”. “Heteroaryl”further encompasses polycyclic ring systems having two or more rings inwhich two or more carbons are common to two adjoining rings (i.e.,“fused rings”) wherein at least one of the rings is heteroaromatic,e.g., the other cyclic ring or rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heterocycles, or combinations thereof. Examples ofheteroaryl rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, furanyl, furazanyl,imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl,octahydroisoquinolinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, pteridinyl,purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined below for “substitutedheteroaryl”.

The term “substituted heteroaryl” refers to a heteroaryl group in whichone or more hydrogen atoms on one or more heteroaromatic rings aresubstituted with one or more substituents including, but not limited to,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxy, carbonyl (such as a ketone, aldehyde, carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, imino,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl,heterocyclyl, alkylaryl, haloalkyl (such as CF₃, —CH₂—CF₃, —CCl₃), —CN,aryl, heteroaryl, and combinations thereof.

The term “substituted alkenyl” refers to alkenyl moieties having one ormore substituents replacing one or more hydrogen atoms on one or morecarbons of the hydrocarbon backbone. Such substituents include, but arenot limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

The term “substituted alkynyl” refers to alkynyl moieties having one ormore substituents replacing one or more hydrogen atoms on one or morecarbons of the hydrocarbon backbone. Such substituents include, but arenot limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl group” is a cycloalkyl groupas defined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as, but not limited to, nitrogen,oxygen, sulphur, or phosphorus.

The term “aralkyl” as used herein is an aryl group having an alkyl,alkynyl, or alkenyl group as defined above attached to the aromaticgroup. An example of an aralkyl group is a benzyl group.

The term “hydroxyalkyl group” as used herein is an alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above that has at least one hydrogenatom substituted with a hydroxyl group.

The term “alkoxyalkyl group” is defined as an alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above that has at least one hydrogen atom substituted with analkoxy group described above.

“Carbonyl,” as used herein, is art-recognized and includes such moietiesas can be represented by the general formula:

wherein X is a bond, or represents an oxygen or a sulfur, and Rrepresents a hydrogen, a substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, —(CH₂)_(m)—R″, or apharmaceutical acceptable salt, R′ represents a hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedalkylaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl or—(CH₂)_(m)—R″; R″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8. Where X is oxygen and R is defines as above, the moiety isalso referred to as a carboxyl group. When X is oxygen and R ishydrogen, the formula represents a ‘carboxylic acid’. Where X is oxygenand R′ is hydrogen, the formula represents a ‘formate’. Where X isoxygen and R or R′ is not hydrogen, the formula represents an “ester”.In general, where the oxygen atom of the above formula is replaced by asulfur atom, the formula represents a ‘thiocarbonyl’ group. Where X issulfur and R or R′ is not hydrogen, the formula represents a‘thioester.’ Where X is sulfur and R is hydrogen, the formula representsa ‘thiocarboxylic acid.’ Where X is sulfur and R′ is hydrogen, theformula represents a ‘thioformate.’ Where X is a bond and R is nothydrogen, the above formula represents a ‘ketone.’ Where X is a bond andR is hydrogen, the above formula represents an ‘aldehyde.’

The term “substituted carbonyl” refers to a carbonyl, as defined above,wherein one or more hydrogen atoms in R, R′ or a group to which themoiety

is attached, are independently substituted. Such substituents include,but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), alkoxyl,phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternizedamino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl,heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “carboxyl” is as defined above for the formula

and is defined more specifically by the formula —R^(iv)COOH, whereinR^(iv) is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,alkylaryl, arylalkyl, aryl, or heteroaryl. In preferred forms, astraight chain or branched chain alkyl, alkenyl, and alkynyl have 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chainalkyl, C₃-C₃₀ for branched chain alkyl, C₂-C₃₀ for straight chainalkenyl and alkynyl, C₃-C₃₀ for branched chain alkenyl and alkynyl),preferably 20 or fewer, more preferably 15 or fewer, most preferably 10or fewer. Likewise, preferred cycloalkyls, heterocyclyls, aryls andheteroaryls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6 or 7 carbons in the ring structure.

The term “substituted carboxyl” refers to a carboxyl, as defined above,wherein one or more hydrogen atoms in R^(iv) are substituted. Suchsubstituents include, but are not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as acarboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester,thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (orquarternized amino), amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,sulfonyl, heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “phenoxy” is art recognized, and refers to a compound of theformula —OR^(v) wherein R is (i.e., —O—C₆H₅). One of skill in the artrecognizes that a phenoxy is a species of the aroxy genus.

The term “substituted phenoxy” refers to a phenoxy group, as definedabove, having one or more substituents replacing one or more hydrogenatoms on one or more carbons of the phenyl ring. Such substituentsinclude, but are not limited to, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), alkoxyl,phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternizedamino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl,heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The terms “aroxy” and “aryloxy,” as used interchangeably herein, arerepresented by —O-aryl or —O-heteroaryl, wherein aryl and heteroaryl areas defined herein.

The terms “substituted aroxy” and “substituted aryloxy,” as usedinterchangeably herein, represent —O-aryl or —O-heteroaryl, having oneor more substituents replacing one or more hydrogen atoms on one or morering atoms of the aryl and heteroaryl, as defined herein. Suchsubstituents include, but are not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as acarboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester,thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (orquarternized amino), amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,sulfonyl, heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. The “alkylthio” moiety is representedby —S-alkyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups having a sulfur radical attached thereto.

The term “substituted alkylthio” refers to an alkylthio group having oneor more substituents replacing one or more hydrogen atoms on one or morecarbon atoms of the alkylthio backbone. Such substituents include, butare not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

The term “phenylthio” is art recognized, and refers to —S—C₆H₅, i.e., aphenyl group attached to a sulfur atom.

The term “substituted phenylthio” refers to a phenylthio group, asdefined above, having one or more substituents replacing a hydrogen onone or more carbons of the phenyl ring. Such substituents include, butare not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

“Arylthio” refers to —S-aryl or —S-heteroaryl groups, wherein aryl andheteroaryl as defined herein.

The term “substituted arylthio” represents —S-aryl or —S-heteroaryl,having one or more substituents replacing a hydrogen atom on one or morering atoms of the aryl and heteroaryl rings as defined herein. Suchsubstituents include, but are not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as acarboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester,thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (orquarternized amino), amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,sulfonyl, heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The terms “amide” or “amido” are used interchangeably, refer to both“unsubstituted amido” and “substituted amido” and are represented by thegeneral formula:

wherein, E is absent, or E is substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aralkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl,wherein independently of E, R and R′ each independently represent ahydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted carbonyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedalkylaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl,—(CH₂)_(m)—R′″, or R and R′ taken together with the N atom to which theyare attached complete a heterocycle having from 3 to 14 atoms in thering structure; R′″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8. In preferred forms, only one of R and R′ can be a carbonyl,e.g., R and R′ together with the nitrogen do not form an imide. Inpreferred forms, R and R′ each independently represent a hydrogen atom,substituted or unsubstituted alkyl, a substituted or unsubstitutedalkenyl, or —(CH₂)_(m)—R′″. When E is oxygen, a carbamate is formed. Thecarbamate cannot be attached to another chemical species, such as toform an oxygen-oxygen bond, or other unstable bonds, as understood byone of ordinary skill in the art.

The term “sulfonyl” is represented by the formula

wherein E is absent, or E is alkyl, alkenyl, alkynyl, aralkyl,alkylaryl, cycloalkyl, aryl, heteroaryl, heterocyclyl, whereinindependently of E, R represents a hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted amine,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, —(CH₂)_(m)—R′″, or E and Rtaken together with the S atom to which they are attached complete aheterocycle having from 3 to 14 atoms in the ring structure; R′″represents a hydroxy group, substituted or unsubstituted carbonyl group,an aryl, a cycloalkyl ring, a cycloalkenyl ring, a heterocycle, or apolycycle; and m is zero or an integer ranging from 1 to 8. In preferredforms, only one of E and R can be substituted or unsubstituted amine, toform a “sulfonamide” or “sulfonamido.” The substituted or unsubstitutedamine is as defined above.

The term “substituted sulfonyl” represents a sulfonyl in which E, R, orboth, are independently substituted. Such substituents include, but arenot limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl,formyl, or an acyl), silyl, ether, ester, thiocarbonyl (such as athioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl,phosphate, phosphonate, phosphinate, amino (or quarternized amino),amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio,sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl,alkylaryl, haloalkyl, —CN, aryl, heteroaryl, and combinations thereof.

The term “sulfonic acid” refers to a sulfonyl, as defined above, whereinR is hydroxyl, and E is absent, or E is substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

The term “sulfate” refers to a sulfonyl, as defined above, wherein E isabsent, oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy,as defined above, and R is independently hydroxyl, alkoxy, aroxy,substituted alkoxy or substituted aroxy, as defined above. When E isoxygen, the sulfate cannot be attached to another chemical species, suchas to form an oxygen-oxygen bond, or other unstable bonds, as understoodby one of ordinary skill in the art.

The term “sulfonate” refers to a sulfonyl, as defined above, wherein Eis oxygen, alkoxy, aroxy, substituted alkoxy or substituted aroxy, asdefined above, and R is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted amine,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, —(CH₂)_(m)—R′″, R′″ representsa hydroxy group, substituted or unsubstituted carbonyl group, an aryl, acycloalkyl ring, a cycloalkenyl ring, a heterocycle, or a polycycle; andm is zero or an integer ranging from 1 to 8. When E is oxygen, sulfonatecannot be attached to another chemical species, such as to form anoxygen-oxygen bond, or other unstable bonds, as understood by one ofordinary skill in the art.

The term “sulfamoyl” refers to a sulfonamide or sulfonamide representedby the formula

wherein E is absent, or E is substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aralkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, whereinindependently of E, R and R′ each independently represent a hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbonyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedalkylaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl,—(CH₂)_(m)—R′″, or R and R′ taken together with the N atom to which theyare attached complete a heterocycle having from 3 to 14 atoms in thering structure; R′″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8. In preferred forms, only one of R and R′ can be a carbonyl,e.g., R and R′ together with the nitrogen do not form an imide.

The term “phosphonyl” is represented by the formula

wherein E is absent, or E is substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted aralkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclyl, wherein,independently of E, R^(vi) and R^(vii) are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbonyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedalkylaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl,—(CH₂)_(m)—R′″, or R and R′ taken together with the P atom to which theyare attached complete a heterocycle having from 3 to 14 atoms in thering structure; R′″ represents a hydroxy group, substituted orunsubstituted carbonyl group, an aryl, a cycloalkyl ring, a cycloalkenylring, a heterocycle, or a polycycle; and m is zero or an integer rangingfrom 1 to 8.

The term “substituted phosphonyl” represents a phosphonyl in which E,R^(vi) and R^(vii) are independently substituted. Such substituentsinclude, but are not limited to, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), alkoxyl,phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternizedamino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl,heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “phosphoryl” defines a phosphonyl in which E is absent, oxygen,alkoxy, aroxy, substituted alkoxy or substituted aroxy, as definedabove, and independently of E, R^(vi) and R^(vii) are independentlyhydroxyl, alkoxy, aroxy, substituted alkoxy or substituted aroxy, asdefined above. When E is oxygen, the phosphoryl cannot be attached toanother chemical species, such as to form an oxygen-oxygen bond, orother unstable bonds, as understood by one of ordinary skill in the art.When E, R^(vi) and R^(vii) are substituted, the substituents include,but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester, thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), alkoxyl,phosphoryl, phosphate, phosphonate, phosphinate, amino (or quarternizedamino), amido, amidine, imine, cyano, nitro, azido, sulfhydryl,alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl,heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “polyaryl” refers to a chemical moiety that includes two ormore aryls, heteroaryls, and combinations thereof. The aryls,heteroaryls, and combinations thereof, are fused, or linked via a singlebond, ether, ester, carbonyl, amide, sulfonyl, sulfonamide, alkyl, azo,and combinations thereof.

The term “substituted polyaryl” refers to a polyaryl in which one ormore of the aryls, heteroaryls are substituted, with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as acarboxyl, alkoxycarbonyl, formyl, or an acyl), silyl, ether, ester,thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino (orquarternized amino), amido, amidine, imine, cyano, nitro, azido,sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido,sulfonyl, heterocyclyl, alkylaryl, haloalkyl, —CN, aryl, heteroaryl, andcombinations thereof.

The term “C₃-C₂₀ cyclic” refers to a substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkenyl, substituted orunsubstituted cycloalkynyl, substituted or unsubstituted heterocyclylthat have from three to 20 carbon atoms, as geometric constraintspermit. The cyclic structures are formed from single or fused ringsystems. The substituted cycloalkyls, cycloalkenyls, cycloalkynyls andheterocyclyls are substituted as defined above for the alkyls, alkenyls,alkynyls and heterocyclyls, respectively.

The term “ether” as used herein is represented by the formula AOA¹,where A and A¹ can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “urethane” as used herein is represented by the formula—OC(O)NRR′, where R and R′ can be, independently, hydrogen, an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above.

The term “silyl group” as used herein is represented by the formula—SiRR′R″, where R, R′, and R″ can be, independently, hydrogen, an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy,or heterocycloalkyl group described above.

The terms “hydroxyl” and “hydroxy” are used interchangeably and arerepresented by —OH.

The terms “thiol” and “sulfhydryl” are used interchangeably and arerepresented by —SH.

The term “oxo” refers to ═O bonded to a carbon atom.

The terms “cyano” and “nitrile” are used interchangeably to refer to—CN.

The term “nitro” refers to —NO₂.

The term “phosphate” refers to —O—PO₃.

The term “azide” or “azido” are used interchangeably to refer to —N₃.

The disclosed compounds and substituent groups, can, independently,possess two or more of the groups listed above. For example, if thecompound or substituent group is a straight chain alkyl group, one ofthe hydrogen atoms of the alkyl group can be substituted with a hydroxylgroup, an alkoxy group, etc. Depending upon the groups that areselected, a first group can be incorporated within second group or,alternatively, the first group can be pendant (i.e., attached) to thesecond group. For example, with the phrase “an alkyl group comprising anester group,” the ester group can be incorporated within the backbone ofthe alkyl group. Alternatively, the ester can be attached to thebackbone of the alkyl group. The nature of the group(s) that is (are)selected will determine if the first group is embedded or attached tothe second group.

The compounds and substituents can be substituted with, independently,with the substituents described above in the definition of“Substituted.”

As used herein, the term “activity” refers to a biological activity.

As used herein, the term “pharmacological activity” refers to theinherent physical properties of a peptide or polypeptide. Theseproperties include but are not limited to half-life, solubility, andstability and other pharmacokinetic properties.

The term “hit” refers to a test compound that shows desired propertiesin an assay. The term “test compound” refers to a chemical to be testedby one or more screening method(s) as a putative modulator. A testcompound can be any chemical, such as an inorganic chemical, an organicchemical, a protein, a peptide, a carbohydrate, a lipid, or acombination thereof. Usually, various predetermined concentrations oftest compounds are used for screening, such as 0.01 micromolar, 1micromolar and 10 micromolar. Test compound controls can include themeasurement of a signal in the absence of the test compound orcomparison to a compound known to modulate the target.

The terms “high,” “higher,” “increases,” “elevates,” or “elevation”refer to increases above basal levels, e.g., as compared to a control.The terms “low,” “lower,” “reduces,” or “reduction” refer to decreasesbelow basal levels, e.g., as compared to a control.

The term “modulate” as used herein refers to the ability of a compoundto change an activity in some measurable way as compared to anappropriate control. As a result of the presence of compounds in theassays, activities can increase or decrease as compared to controls inthe absence of these compounds. Preferably, an increase in activity isat least 25%, more preferably at least 50%, most preferably at least100% compared to the level of activity in the absence of the compound.Similarly, a decrease in activity is preferably at least 25%, morepreferably at least 50%, most preferably at least 100% compared to thelevel of activity in the absence of the compound. A compound thatincreases a known activity is an “agonist”. One that decreases, orprevents, a known activity is an “antagonist”.

The term “inhibit” means to reduce or decrease in activity orexpression. This can be a complete inhibition or activity or expression,or a partial inhibition. Inhibition can be compared to a control or to astandard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disease or conditions so asto prevent a physical manifestation of aberrations associated with thedisease or condition.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, or individualin the case of humans; veterinarian in the case of animals, includingnon-human mammals) that a subject requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a care giver's expertise, but that includes theknowledge that the subject is ill, or will be ill, as the result of acondition that is treatable by the disclosed compounds.

As used herein, “subject” includes, but is not limited to, animals,plants, bacteria, viruses, parasites and any other organism or entity.The subject can be a vertebrate, more specifically a mammal (e.g., ahuman, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow,cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.The subject can be an invertebrate, more specifically an arthropod(e.g., insects and crustaceans). The term does not denote a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are intended to be covered. A patient refers toa subject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects.

By “treatment” and “treating” is meant the medical management of asubject with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. It is understood that treatment, while intendedto cure, ameliorate, stabilize, or prevent a disease, pathologicalcondition, or disorder, need not actually result in the cure,ameliorization, stabilization or prevention. The effects of treatmentcan be measured or assessed as described herein and as known in the artas is suitable for the disease, pathological condition, or disorderinvolved. Such measurements and assessments can be made in qualitativeand/or quantitative terms. Thus, for example, characteristics orfeatures of a disease, pathological condition, or disorder and/orsymptoms of a disease, pathological condition, or disorder can bereduced to any effect or to any amount.

A cell can be in vitro. Alternatively, a cell can be in vivo and can befound in a subject. A “cell” can be a cell from any organism including,but not limited to, a bacterium.

By the term “effective amount” of a compound as provided herein is meanta nontoxic but sufficient amount of the compound to provide the desiredresult.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to a subject along with the selected compound withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

II. Compositions

An unbiased strategy in which a high-throughput chemical biology screenwas employed to identify small molecule probes that influence downstreampathways involved in the control of cellular neutral lipid stores. Onesuch molecule, SBI-477, coordinately reduced myocyte lipid stores andincreased glucose uptake.

SBI-477 is a potent inhibitor of fatty acid incorporation intotriglyceride in human skeletal myocytes. In parallel, SBI-477 increasesmyocyte glucose uptake by activating insulin signaling. The cellularactions of SBI-477 are attributable, at least in part, to inhibition ofthe transcription factor MondoA resulting in reduced expression of TAGsynthesis genes and suppressed transcription of genes encodingsuppressors of insulin signaling. An analog of SBI-477, named SBI-993was used, which exhibited improved potency and suitable pharmacokineticproperties for in vivo bioavailability.

Accordingly, in one aspect the compositions disclosed herein includeMondo transcription factor inhibitors (herein, “Mondo inhibitors”),preferably, MondoA and/or MondoB inhibitors. Mondo inhibitors aredefined herein generally as compounds and/or molecules that reduce theexpression, amount, or activity of at least one Mondo family protein,for example, the transcription factors, MondoA or MondoB cells andparticularly, in the nucleus. A MondoA (or MondoB) inhibitor isconsidered to inhibit the activity of MondoA (or MondoB) if, forexample, it reduces nuclear localization/levels of MondoA in cellstreated with the inhibitor, when compared to non-treated cells. Theinhibitor can reduce nuclear localization of MondoA (or MondoB) by forexample, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. These include butare not limited to small molecule compounds as further described below,proteins, peptides and inhibitory nucleic acid molecules.

A. Compounds

The compositions disclosed herein include SBI-477 and its analogs(collectively, SBI-477 compounds) and/or protein/peptide/nucleic acidMondo inhibitors. In a preferred embodiment, the Mondo inhibitor is aninhibitor of MondoA and/or MondoB.

1. Small Molecule Compounds

In one aspect described herein are compounds having the Formula I.

A-L₁-B-L₂-D-(R₂′)_(n),   Formula I

wherein

A is substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstitutedC₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio;

L₁ is —C(O)NR′—, —NR′C(O)—, —C(O)O—, —OC(O)—, —O—, a bond, substitutedalkyl, unsubstituted alkyl, substituted alkylene, unsubstitutedalkylene, substituted alkenyl, unsubstituted alkenyl, substitutedalkynyl, unsubstituted alkynyl, substituted alkoxy, unsubstitutedalkoxy, substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, substituted alkylthio, or unsubstitutedalkylthio;

L₂ is —C(O)NR′—, —NR′C(O)—, —C(O)O—, —OC(O)—, —O—, absent, a bond,substituted alkyl, unsubstituted alkyl, substituted alkylene,unsubstituted alkylene, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,or unsubstituted alkylthio;

R′ is, for each occurrence, independently hydrogen, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl,substituted heterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,or unsubstituted alkylthio;

B and D are independently substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, substituted alkylthio, or unsubstitutedalkylthio, wherein if L₂ is absent and B and D are each independentlysubstituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, B and D are a fused ring or a polycyclicsystem;

R₂′ is, for each occurrence, independently hydrogen, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl,substituted heterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl, nitro, cyano,or two R2′ units fuse to form substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,or unsubstituted heterocyclyl, and

wherein n is an integer between 1 and 5, inclusive.

In some forms, the compounds have the general Formula I, as describedabove, with the exception that B and D are independently substitutedaryl, unsubstituted aryl, substituted heteroaryl, or unsubstitutedheteroaryl.

In some forms the compounds of Formula I are represented by the generalFormula II.

wherein

A is as defined above for Formula I,

L₁ is —C(O)NR′—, —NR′C(O)—, —C(O)O—, —OC(O)—, —O—, a bond, substitutedalkyl, unsubstituted alkyl, substituted alkylene, unsubstitutedalkylene, substituted alkenyl, unsubstituted alkenyl, substitutedalkynyl, unsubstituted alkynyl, substituted alkoxy, unsubstitutedalkoxy, substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, substituted alkylthio, or unsubstitutedalkylthio;

L₂ is —C(O)NR′—, —NR′C(O)—, —C(O)O—, —OC(O)—, —O—, absent, a bond,substituted alkyl, unsubstituted alkyl, substituted alkylene,unsubstituted alkylene, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,or unsubstituted alkylthio;

R′ is hydrogen, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl,unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstitutedheterocyclyl, substituted alkyl, unsubstituted alkyl, substitutedalkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstitutedalkynyl, substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio;

R₁′, R₂′, R₃′, R₄′ and R₅′ are independently hydrogen, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl,substituted heterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, or cyano, or any two adjacent R₁′, R₂′, R₃′, R₄′ andR₅′ fuse to form substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl,unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl, orunsubstituted heterocyclyl, and

X and Y are, as valence permits, independently C, O, N, S, CR₆′R₇′, orNR₈′, wherein R₆′, R₇′, and R₈′ are independently hydrogen, substitutedaryl, unsubstituted aryl, substituted heteroaryl, unsubstitutedheteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio.

In some forms, the compounds of Formula II are represented by thegeneral Formula III:

wherein L₁, L₂, X, Y, R₁′, R₂, R₃, R₄′ and R₅′ are as described abovefor formula II,

R₉′, R₁₀′, R₁₁′, R₁₂′, R₁₃′, and R₁₄′ are independently C, O, N, S,wherein the bonds between adjacent R₉′ to R₁₄′ are double or singleaccording to valency, wherein R₉′ to R₁₄′ are bound to none, one, or twohydrogens according to valency,

wherein R₁₅′ is, for each occurrence, independently hydrogen,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstitutedC₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,amide, substituted amide, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, substituted alkylthio, unsubstituted alkylthio,halogen (F, Cl, Br, I), hydroxyl, nitro, or cyano, and

m is an integer between 1 and 12, inclusive.

In some forms, the compounds of Formula III are represented by thegeneral Formula IV or general Formula V:

wherein L₁, L₂, X, Y, R₁′, R₂′, R₃, R₄′ and R₅′ are as described abovefor Formula III,

R₁₆′, R₁₇′, R₁₈′, R₁₉′, R₂₀′, R₂₁′, R₂₂′, R₂₃′, R₂₄′, and R₂₅′ areindependently hydrogen, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, or cyano.

In some forms, the compounds of Formula I are represented by the generalFormula VI or general Formula VII:

wherein L₁, L₂, X, Y, R₁′, R₂′, R₃′, R₄′ and R₅′ are as described abovefor Formula III,

R₁₆′, R₁₇′, R₁₈′, R₁₉′, R₂₀′, R₂₁′, R₂₂′, R₂₃′, R₂₄′, and R₂₅′ areindependently hydrogen, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, or cyano.

In some forms, the compounds of Formula IV are represented by FormulaVIII,

wherein

L₁ is —NR′C(O)—, —C(O)NR′—, or substituted amino,

R₁′, R₂′ and R₃′ are independently hydrogen, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, cyano, R₂′ and R₃′ combine to form, substituted heterocyclyl orunsubstituted heterocyclyl, and

R₁₈′ and R₁₉′ are independently hydrogen, substituted alkoxy,unsubstituted alkoxy, substituted amino, or unsubstituted amino.

In some forms, the compounds of Formula II are represented by FormulaIX:

wherein

R₁′, R₂′ and R₃′ are independently hydrogen, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, cyano, R₁′ and R₂′, or R₂′ and R₃′ combine to form substitutedheterocyclyl or unsubstituted heterocyclyl, and

A is a substituted alkyl, unsubstituted alkyl, substituted C₃-C₃₀cycloalkyl, or unsubstituted C₃-C₃₀ cycloalkyl.

In some forms, the compounds of Formula VI are represented by thegeneral Formula X,

wherein

R₂′ is hydrogen, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,amide, substituted amide, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, substituted alkylthio, unsubstituted alkylthio,halogen (F, Cl, Br, I), hydroxyl, nitro, cyano, and

R₁₈′ and R₁₉′ are independently substituted alkoxy, or unsubstitutedalkoxy.

In some forms, the compounds have the general Formula XI

R₁′, R₂, R₃, R₄′, and R₅′ are independently hydrogen, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, and

R₁₈′ and R₁₉′ are independently substituted alkoxy, or unsubstitutedalkoxy.

In some forms, the compounds of Formula IV have the general Formula XII,

wherein

L₂ is a bond, substituted alkylene, or unsubstituted alkylene,

R₁′, R₂, R₃, R₄′, R₅′, and R₂₅′ are independently hydrogen, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, and

R₁₈′ and R₁₉′ are independently substituted alkoxy, or unsubstitutedalkoxy.

Specific compounds (Cpd) prepared according to General Synthetic SchemeI are shown below.

Exemplary compounds (Cpd) prepared according to General Synthetic Scheme2 are shown below.

Exemplary compounds (Cpd) prepared according to General Synthetic Scheme3 are shown below.

Example 75

Exemplary compounds (Cpd) prepared according to General Synthetic Scheme4 are shown below.

Example 79

Exemplary compounds (Cpd) prepared according to General Synthetic Scheme5 are shown below.

Exemplary compounds (Cpd) prepared according to General Synthetic Scheme6 are shown below.

Every compound within the above definition is intended to be and shouldbe considered to be specifically disclosed herein. Further, everysubgroup that can be identified within the above definition is intendedto be and should be considered to be specifically disclosed herein. As aresult, it is specifically contemplated that any compound, or subgroupof compounds can be either specifically included for or excluded fromuse or included in or excluded from a list of compounds. For example,any one or more of the compounds described herein, with a structuredepicted herein, or referred to in the Tables or the Examples herein canbe specifically included, excluded, or combined in any combination, in aset or subgroup of such compounds. Such specific sets, subgroups,inclusions, and exclusions can be applied to any aspect of thecompositions and methods described here. For example, a set of compoundsthat specifically excludes one or more particular compounds can be usedor applied in the context of compounds per se (for example, a list orset of compounds), compositions including the compound (including, forexample, pharmaceutical compositions), any one or more of the disclosedmethods, or combinations of these. Different sets and subgroups ofcompounds with such specific inclusions and exclusions can be used orapplied in the context of compounds per se, compositions including oneor more of the compounds, or any of the disclosed methods. All of thesedifferent sets and subgroups of compounds—and the different sets ofcompounds, compositions, and methods using or applying the compounds—arespecifically and individual contemplated and should be considered asspecifically and individually described. As an example, compound 7,compound 38, and compound 82 can be specifically included or excluded,as a group or individually, from any compounds per se (for example, alist or set of compounds), compositions including the compound(including, for example, pharmaceutical compositions), or any one ormore of the disclosed methods, or combinations of these.

The compounds represented by Formula I can be optically active orracemic.

2. Salts and Derivatives

Also described herein are pharmaceutically acceptable nontoxic ester,amide, and salt derivatives of those compounds of formula (I) containinga carboxylic acid moiety.

Formula I also encompasses pharmaceutically acceptable salts.Pharmaceutically acceptable salts are prepared by treating the free acidwith an appropriate amount of a pharmaceutically acceptable base.Representative pharmaceutically acceptable bases are ammonium hydroxide,sodium hydroxide, potassium hydroxide, lithium hydroxide, calciumhydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide,copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine, and the like. In one aspect, the reaction isconducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C. such as at room temperature. The molar ratio of compoundsof structural formula (I) to base used are chosen to provide the ratiodesired for any particular salts. For preparing, for example, theammonium salts of the free acid starting material, the starting materialcan be treated with approximately one equivalent of pharmaceuticallyacceptable base to yield a neutral salt.

Ester derivatives are typically prepared as precursors to the acid formof the compounds—as illustrated in the examples below—and accordinglycan serve as prodrugs. Generally, these derivatives will be lower alkylesters such as methyl, ethyl, and the like. Amide derivatives —(CO)NH₂,—(CO)NHR and —(CO)NR₂, where R is an alkyl group defined above, can beprepared by reaction of the carboxylic acid-containing compound withammonia or a substituted amine.

B. Protein/Peptide/Nucleic Acid Mondo Inhibitors

The disclosed composition can also include a Mondo family transcriptionfactor inhibitor (herein, a Mondo inhibitor) which is a protein, peptideor nucleic acid molecule. In a preferred embodiment the familytranscription factor is MondoA and/or MondoB (ChREBP). MondoA, alsoknown as MLXIP (MLX interacting protein), KIAA0867 or MIR, is a 919amino acid protein that localizes to the nucleus and the cytoplasm, aswell as to the outer mitochondrial membrane, and contains one bHLHdomain. Expressed in a variety of tissues with highest expression inskeletal muscle, MondoA functions as a dimeric structure that binds DNAat the canonical E box sequence 5′-CACGTG-3′ and is involved intranscriptional activation and glucose-responsive gene regulation.Multiple isoforms of MondoA exist due to alternative splicing events(UniprotKB identifier Q9HAP2 (MLXIP_HUMAN) which includes 5 isoforms:Q9HAP2-1 (the “canonical sequence”); Q9HAP2-2; Q9HAP2-3; Q9HAP2-4 orQ9HAP2-5 (Billin, et al., Mol. Cell. Biol. 20:8845-8854 (2000); Nagase,et al., DNA Res. 5:355-364 (1998); Genome Res. 14:2121-2127 (2004)).

Nucleic acid molecules that inhibit expression of a gene or nucleic acidcan be referred to as “inhibitory nucleic acid” (referring to theircomposition). Inhibitory nucleic acid technologies are known in the artand include, but are not limited to, antisense oligonucleotides,catalytic nucleic acids such as ribozymes and deoxyribozymes, aptamers,triplex forming nucleic acids, external guide sequences, and RNAinterference molecules (RNAi), particularly small nucleic acidmolecules, such as short interfering nucleic acid (s1NA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA),and short hairpin RNA (shRNA) molecules capable of mediating RNAinterference (RNAi).

An inhibitory nucleic acid can reduce expression of a protein having theamino acid sequence of MondoA disclosed under UniprotKB accession no.Q9HAP2 (MLXIP_HUMAN) (including Q9HAP2-1; Q9HAP2-2; Q9HAP2-3; Q9HAP2-4or Q9HAP2-5;) or a variant thereof, collectively, “MondoA proteins”) orChREBP (MondoB) disclosed UniprotKB accession no. Q9NP71 (which includealternative splice variants disclosed under Q9NP71-1 (“canonical”sequence); Q9NP71-2; Q9NP71-3; Q9NP71-4; Q9NP71-5; or Q9NP71-6) or avariant thereof, collectively, “MondoB proteins”. The inhibitory nucleicacid can reduce expression of an mRNA sequence encoding a MondoA orMondoB protein or genomic DNA encoding the mRNA.

i. Inhibitory Nucleic Acids

The expression or amount of MondoA (and/or MondoB) can be reduced insome cases using RNA interference, whereby double-stranded RNA (dsRNA,also referred to herein as siRNAs or ds siRNAs, for double-strandedsmall interfering RNAs) induces the sequence-specific degradation oftargeted mRNA in cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev.:12, 225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). In mammaliancells, RNAi can be triggered by 21-nucleotide (nt) duplexes of smallinterfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561 (2002);Elbashir et al., Nature 411:494-498 (2001)), or by micro-RNAs (miRNA),functional small-hairpin RNA (shRNA), or other dsRNAs which can beexpressed in vivo using DNA templates with RNA polymerase III promoters(Zeng et al., Mol. Cell 9:1327-1333 (2002); Paddison et al., Genes Dev.16:948-958 (2002); Lee et al., Nature Biotechnol. 20:500-505 (2002);Paul et al., Nature Biotechnol. 20:505-508 (2002); Tuschl, T., NatureBiotechnol. 20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci. USA99(9):6047-6052 (2002); McManus et al., RNA 8:842-850 (2002); Sui etal., Proc. Natl. Acad. Sci. USA 99(6):5515-5520 (2002)).

In a preferred embodiment, the inhibitory nucleic acid is an siRNA. Inone embodiment, the inhibitory nucleic acid has 100% sequence identitywith at least a part of the target mRNA. However, inhibitory nucleicacids having 70%, 80% or greater than 90% or 95% sequence identity maybe used. Thus sequence variations that might be expected due to geneticmutation, strain polymorphism, or evolutionary divergence can betolerated. siRNA specific for MondoA and MondoB are commerciallyavailable. For example, MondoA siRNA (h) and ChREBP siRNA (h), each apool of 3 target-specific 19-25 nt siRNAs designed to knock down geneexpression of MondoA and MondoB respectively, is available from SantaCruz Biotechnology, Inc. In addition, there are a number of companiesthat will generate interfering RNAs for a specific gene. Thermo ElectronCorporation (Waltham, Mass.) has launched a custom synthesis service forsynthetic short interfering RNA (siRNA). Each strand is composed of18-20 RNA bases and two DNA bases overhang on the 3′ terminus.Dharmacon, Inc. (Lafayette, Colo.) provides siRNA duplexes using the2′-ACE RNA synthesis technology. Qiagen (Valencia, Calif.) usesTOM-chemistry to offer siRNA with high individual coupling yields (L₁,et al., Nat. Med., 11(9):944-951 (2005).

In some forms the Mondo inhibitor is an antisense oligonucleotide. An“antisense” nucleic acid sequence (antisense oligonucleotide) caninclude a nucleotide sequence that is complementary to a “sense” nucleicacid encoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to the Mondo or MondoBmRNA. Antisense nucleic acid sequences and delivery methods are wellknown in the art (Goodchild, Curr. Opin. Mol. Ther., 6(2):120-128(2004); Clawson, et al., Gene Ther., 11(17):1331-1341 (2004)), which areincorporated herein by reference in their entirety. An antisense nucleicacid can be constructed using chemical synthesis and enzymatic ligationreactions using procedures known in the art. For example, an antisensenucleic acid (e.g., an antisense oligonucleotide) can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used. Theantisense nucleic acid also can be produced biologically using anexpression vector into which a nucleic acid has been subcloned in anantisense orientation (i.e., RNA transcribed from the inserted nucleicacid will be of an antisense orientation to a target nucleic acid ofinterest, described further in the following subsection).

In some forms the Mondo inhibitor is a ribozyme specific for a Mondofamily transcription factor. Ribozymes are a type of RNA that can beengineered to enzymatically cleave and inactivate other RNA targets in aspecific, sequence-dependent fashion. Ribozymes and methods for theirdelivery are well known in the art (Hendry, et al., BMC Chem. Biol.,4(1):1 (2004); Grassi, et al., Curr. Pharm. Biotechnol., 5(4):369-386(2004); Bagheri, et al., Curr. Mol. Med., 4(5):489-506 (2004);Kashani-Sabet M., Expert Opin. Biol. Ther., 4(11):1749-1755 (2004), eachof which are incorporated herein by reference in its entirety. Bycleaving the target RNA, ribozymes inhibit translation, thus preventingthe expression of the target gene. Ribozymes can be chemicallysynthesized in the laboratory and structurally modified to increasetheir stability and catalytic activity using methods known in the art.Alternatively, ribozyme genes can be introduced into cells throughgene-delivery mechanisms known in the art.

In some forms, the Mondo inhibitor is a Triplex forming nucleic acid.Triplex forming nucleic acid molecules are molecules that can interactwith either double-stranded or single-stranded nucleic acid. Whentriplex molecules interact with a target region, a structure called atriplex is formed, in which there are three strands of DNA forming acomplex dependent on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a K_(d) less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹². Examples of how to make and use triplex formingmolecules to bind a variety of different target molecules are known inthe art.

In some forms, the Mondo inhibitor is an external guide sequences(EGSs). EGSs are molecules that bind a target nucleic acid moleculeforming a complex, and this complex is recognized by RNase P, whichcleaves the target molecule. EGSs can be designed to specifically targeta RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA)within a cell. Bacterial RNAse P can be recruited to cleave virtuallyany RNA sequence by using an EGS that causes the target RNA:EGS complexto mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAseP-directed cleavage of RNA can be utilized to cleave desired targetswithin eukaryotic cells. Examples of how to make and use EGS moleculesto facilitate cleavage of a variety of different target molecules areknown in the art.

ii. Antibodies

In some forms, the Mondo inhibitor can be an antibody. For example, theMondo inhibitor can be an anti-MondoA or anti-MondoB antibody. Forexample, an anti-MondoA (or MondoB) antibody can be an antibody specificfor MondoA (or MondoB), preferably, a monoclonal antibody. A monoclonalantibody composition is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone type of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. Such antibodies were first described by Kohler and Milstein,Nature, 1975, 256:495-497, the disclosure of which is hereinincorporated by reference. An exemplary hybridoma technology isdescribed by Niman et al., Proc. Natl. Acad. Sci. U.S.A., 1983,80:4949-4953. Other methods of producing monoclonal antibodies, ahybridoma cell, or a hybridoma cell culture are also well known. See forexample, Antibodies: A Laboratory Manual, Harlow et al., Cold SpringHarbor Laboratory, 1988; or the method of isolating monoclonalantibodies from an immunological repertoire as described by Sasatry, etal., Proc. Natl. Acad. Sci. USA, 1989, 86:5728-5732; and Huse et al.,Science, 1981, 246:1275-1281.

iii. Aptamers

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules as well as large molecules, such as reverse transcriptase.Aptamers can bind very tightly with K_(d)'s from the target molecule ofless than 10-12 M. It is preferred that the aptamers bind the targetmolecule with a K_(d) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamerscan bind the target molecule with a very high degree of specificity. Forexample, aptamers have been isolated that have greater than a 10,000fold difference in binding affinities between the target molecule andanother molecule that differ at only a single position on the molecule.It is preferred that the aptamer have a K_(d) with the target moleculeat least 10, 100, 1000, 10,000, or 100,000 fold lower than the K_(d)with a background binding molecule. It is preferred when doing thecomparison for a polypeptide for example, that the background moleculebe a different polypeptide. Representative examples of how to make anduse aptamers to bind a variety of different target molecules are knownin the art.

C. Formulations

The SBI-477 compounds and/or Mondo inhibitors described herein can beformulated for enteral, parenteral, topical, or pulmonaryadministration. The compounds can be combined with one or morepharmaceutically acceptable carriers and/or excipients that areconsidered safe and effective and may be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. See, e.g., Remington's Pharmaceutical Sciences, latestedition, by E.W. Martin Mack Pub. Co., Easton, Pa., which disclosestypical carriers and conventional methods of preparing pharmaceuticalcompositions that can be used in conjunction with the preparation offormulations of the compounds described herein and which is incorporatedby reference herein. These most typically would be standard carriers foradministration of compositions to humans. In one aspect, humans andnon-humans, including solutions such as sterile water, saline, andbuffered solutions at physiological pH. Other compounds will beadministered according to standard procedures used by those skilled inthe art.

These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

1. Parenteral Formulations

The compounds described herein can be formulated for parenteraladministration. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intravitreally, intratumorally, intramuscularly, subcutaneously,subconjunctivally, intravesicularly, intrapericardially,intraumbilically, by injection, and by infusion.

Parenteral formulations can be prepared as aqueous compositions usingtechniques known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

If for intravenous administration, the compositions are packaged insolutions of sterile isotonic aqueous buffer. Where necessary, thecomposition may also include a solubilizing agent. The components of thecomposition are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or concentratedsolution in a hermetically sealed container such as an ampoule or sachetindicating the amount of active agent. If the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile water orsaline can be provided so that the ingredients may be mixed prior toinjection.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the active compounds as the free acid orbase or pharmacologically acceptable salts thereof can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents,viscosity modifying agents, and combination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface-active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene, and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-.beta.-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water-soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

(a) Controlled Release Formulations

The parenteral formulations described herein can be formulated forcontrolled release including immediate release, delayed release,extended release, pulsatile release, and combinations thereof.

1. Nano- and Microparticles

For parenteral administration, the one or more compounds, and optionalone or more additional active agents, can be incorporated intomicroparticles, nanoparticles, or combinations thereof that providecontrolled release of the compounds and/or one or more additional activeagents. In forms wherein the formulations contains two or more drugs,the drugs can be formulated for the same type of controlled release(e.g., delayed, extended, immediate, or pulsatile) or the drugs can beindependently formulated for different types of release (e.g., immediateand delayed, immediate and extended, delayed and extended, delayed andpulsatile, etc.).

For example, the compounds and/or one or more additional active agentscan be incorporated into polymeric microparticles, which providecontrolled release of the drug(s). Release of the drug(s) is controlledby diffusion of the drug(s) out of the microparticles and/or degradationof the polymeric particles by hydrolysis and/or enzymatic degradation.Suitable polymers include ethylcellulose and other natural or syntheticcellulose derivatives.

Polymers, which are slowly soluble and form a gel in an aqueousenvironment, such as hydroxypropyl methylcellulose or polyethyleneoxide, can also be suitable as materials for drug containingmicroparticles. Other polymers include, but are not limited to,polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such aspolylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)(PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof,poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactoneand copolymers thereof, and combinations thereof.

Alternatively, the drug(s) can be incorporated into microparticlesprepared from materials which are insoluble in aqueous solution orslowly soluble in aqueous solution, but are capable of degrading withinthe GI tract by means including enzymatic degradation, surfactant actionof bile acids, and/or mechanical erosion. As used herein, the term“slowly soluble in water” refers to materials that are not dissolved inwater within a period of 30 minutes. Preferred examples include fats,fatty substances, waxes, wax-like substances and mixtures thereof.Suitable fats and fatty substances include fatty alcohols (such aslauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids andderivatives, including but not limited to fatty acid esters, fatty acidglycerides (mono-, di- and tri-glycerides), and hydrogenated fats.Specific examples include, but are not limited to hydrogenated vegetableoil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenatedoils available under the trade name Sterotex®, stearic acid, cocoabutter, and stearyl alcohol. Suitable waxes and wax-like materialsinclude natural or synthetic waxes, hydrocarbons, and normal waxes.Specific examples of waxes include beeswax, glycowax, castor wax,carnauba wax, paraffins and candelilla wax. As used herein, a wax-likematerial is defined as any material, which is normally solid at roomtemperature and has a melting point of from about 30 to 300° C.

In some cases, it may be desirable to alter the rate of waterpenetration into the microparticles. To this end, rate-controlling(wicking) agents can be formulated along with the fats or waxes listedabove. Examples of rate-controlling materials include certain starchderivatives (e.g., waxy maltodextrin and drum dried corn starch),cellulose derivatives (e.g., hydroxypropylmethyl-cellulose,hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose),alginic acid, lactose and talc. Additionally, a pharmaceuticallyacceptable surfactant (for example, lecithin) may be added to facilitatethe degradation of such microparticles.

Proteins, which are water insoluble, such as zein, can also be used asmaterials for the formation of drug containing microparticles.Additionally, proteins, polysaccharides and combinations thereof, whichare water-soluble, can be formulated with drug into microparticles andsubsequently cross-linked to form an insoluble network. For example,cyclodextrins can be complexed with individual drug molecules andsubsequently cross-linked.

2. Method of Making Nano- and Microparticles

Encapsulation or incorporation of drug into carrier materials to producedrug-containing microparticles can be achieved through knownpharmaceutical formulation techniques. In the case of formulation infats, waxes or wax-like materials, the carrier material is typicallyheated above its melting temperature and the drug is added to form amixture comprising drug particles suspended in the carrier material,drug dissolved in the carrier material, or a mixture thereof.Microparticles can be subsequently formulated through several methodsincluding, but not limited to, the processes of congealing, extrusion,spray chilling or aqueous dispersion. In a preferred process, wax isheated above its melting temperature, drug is added, and the moltenwax-drug mixture is congealed under constant stirring as the mixturecools. Alternatively, the molten wax-drug mixture can be extruded andspheronized to form pellets or beads. These processes are known in theart.

For some carrier materials it may be desirable to use a solventevaporation technique to produce drug-containing microparticles. In thiscase drug and carrier material are co-dissolved in a mutual solvent andmicroparticles can subsequently be produced by several techniquesincluding, but not limited to, forming an emulsion in water or otherappropriate media, spray drying or by evaporating off the solvent fromthe bulk solution and milling the resulting material.

In some forms, drug in a particulate form is homogeneously dispersed ina water-insoluble or slowly water soluble material. To minimize the sizeof the drug particles within the composition, the drug powder itself maybe milled to generate fine particles prior to formulation. The processof jet milling, known in the pharmaceutical art, can be used for thispurpose. In some forms, drug in a particulate form is homogeneouslydispersed in a wax or wax like substance by heating the wax or wax likesubstance above its melting point and adding the drug particles whilestirring the mixture. In this case a pharmaceutically acceptablesurfactant may be added to the mixture to facilitate the dispersion ofthe drug particles.

The particles can also be coated with one or more modified releasecoatings. Solid esters of fatty acids, which are hydrolyzed by lipases,can be spray coated onto microparticles or drug particles. Zein is anexample of a naturally water-insoluble protein. It can be coated ontodrug containing microparticles or drug particles by spray coating or bywet granulation techniques. In addition to naturally water-insolublematerials, some substrates of digestive enzymes can be treated withcross-linking procedures, resulting in the formation of non-solublenetworks. Many methods of cross-linking proteins, initiated by bothchemical and physical means, have been reported. One of the most commonmethods to obtain cross-linking is the use of chemical cross-linkingagents. Examples of chemical cross-linking agents include aldehydes(gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, andgenipin. In addition to these cross-linking agents, oxidized and nativesugars have been used to cross-link gelatin. Cross-linking can also beaccomplished using enzymatic means; for example, transglutaminase hasbeen approved as a GRAS substance for cross-linking seafood products.Finally, cross-linking can be initiated by physical means such asthermal treatment, UV irradiation and gamma irradiation.

To produce a coating layer of cross-linked protein surrounding drugcontaining microparticles or drug particles, a water-soluble protein canbe spray coated onto the microparticles and subsequently cross-linked bythe one of the methods described above. Alternatively, drug-containingmicroparticles can be microencapsulated within protein bycoacervation-phase separation (for example, by the addition of salts)and subsequently cross-linked. Some suitable proteins for this purposeinclude gelatin, albumin, casein, and gluten.

Polysaccharides can also be cross-linked to form a water-insolublenetwork. For many polysaccharides, this can be accomplished by reactionwith calcium salts or multivalent cations, which cross-link the mainpolymer chains. Pectin, alginate, dextran, amylose and guar gum aresubject to cross-linking in the presence of multivalent cations.Complexes between oppositely charged polysaccharides can also be formed;pectin and chitosan, for example, can be complexed via electrostaticinteractions.

(b) Injectable/Implantable Formulations

The compounds described herein can be incorporated intoinjectable/implantable solid or semi-solid implants, such as polymericimplants. In some forms, the compounds are incorporated into a polymerthat is a liquid or paste at room temperature, but upon contact withaqueous medium, such as physiological fluids, exhibits an increase inviscosity to form a semi-solid or solid material. Exemplary polymersinclude, but are not limited to, hydroxyalkanoic acid polyesters derivedfrom the copolymerization of at least one unsaturated hydroxy fatty acidcopolymerized with hydroxyalkanoic acids. The polymer can be melted,mixed with the active substance and cast or injection molded into adevice. Such melt fabrication requires polymers having a melting pointthat is below the temperature at which the substance to be delivered andpolymer degrade or become reactive. The device can also be prepared bysolvent casting where the polymer is dissolved in a solvent and the drugdissolved or dispersed in the polymer solution and the solvent is thenevaporated. Solvent processes require that the polymer be soluble inorganic solvents. Another method is compression molding of a mixedpowder of the polymer and the drug or polymer particles loaded with theactive agent.

Alternatively, the compounds can be incorporated into a polymer matrixand molded, compressed, or extruded into a device that is a solid atroom temperature. For example, the compounds can be incorporated into abiodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids(PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides,polyorthoesters, polyphosphazenes, proteins and polysaccharides such ascollagen, hyaluronic acid, albumin and gelatin, and combinations thereofand compressed into solid device, such as disks, or extruded into adevice, such as rods.

The release of the one or more compounds from the implant can be variedby selection of the polymer, the molecular weight of the polymer, and/ormodification of the polymer to increase degradation, such as theformation of pores and/or incorporation of hydrolyzable linkages.Methods for modifying the properties of biodegradable polymers to varythe release profile of the compounds from the implant are well known inthe art.

2. Enteral Formulations

Oral formulations can include standard carriers such as pharmaceuticalgrades of mannitol, lactose, sodium saccharine, starch, magnesiumstearate, cellulose, magnesium carbonate, etc. Such compositions willcontain a therapeutically effective amount of the compound and/orantibiotic together with a suitable amount of carrier so as to providethe proper form to the patient based on the mode of administration to beused. Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations may be prepared using a pharmaceutically acceptablecarrier. As generally used herein “carrier” includes, but is not limitedto, diluents, preservatives, binders, lubricants, disintegrators,swelling agents, fillers, stabilizers, and combinations thereof.

Carrier also includes all components of the coating composition, whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

“Diluents”, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

“Binders” are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

“Lubricants” are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

“Disintegrants” are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

“Stabilizers” are used to inhibit or retard drug decompositionreactions, which include, by way of example, oxidative reactions.Suitable stabilizers include, but are not limited to, antioxidants,butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters;Vitamin E, tocopherol and its salts; sulfites such as sodiummetabisulphite; cysteine and its derivatives; citric acid; propylgallate, and butylated hydroxyanisole (BHA).

(a) Controlled Release Enteral Formulations

Oral dosage forms, such as capsules, tablets, solutions, andsuspensions, can for formulated for controlled release. For example, theone or more compounds and optional one or more additional active agentscan be formulated into nanoparticles, microparticles, and combinationsthereof, and encapsulated in a soft or hard gelatin or non-gelatincapsule or dispersed in a dispersing medium to form an oral suspensionor syrup. The particles can be formed of the drug and a controlledrelease polymer or matrix. Alternatively, the drug particles can becoated with one or more controlled release coatings prior toincorporation in to the finished dosage form.

In another form, the one or more compounds and optional one or moreadditional active agents are dispersed in a matrix material, which gelsor emulsifies upon contact with an aqueous medium, such as physiologicalfluids. In the case of gels, the matrix swells entrapping the activeagents, which are released slowly over time by diffusion and/ordegradation of the matrix material. Such matrices can be formulated astablets or as fill materials for hard and soft capsules.

In still another form, the one or more compounds, and optional one ormore additional active agents are formulated into a sold oral dosageform, such as a tablet or capsule, and the solid dosage form is coatedwith one or more controlled release coatings, such as a delayed releasecoatings or extended release coatings. The coating or coatings may alsocontain the compounds and/or additional active agents.

(1) Extended Release Dosage Forms

The extended release formulations are generally prepared as diffusion orosmotic systems, which are known in the art. A diffusion systemtypically consists of two types of devices, a reservoir and a matrix,and is well known and described in the art. The matrix devices aregenerally prepared by compressing the drug with a slowly dissolvingpolymer carrier into a tablet form. The three major types of materialsused in the preparation of matrix devices are insoluble plastics,hydrophilic polymers, and fatty compounds. Plastic matrices include, butare not limited to, methyl acrylate-methyl methacrylate, polyvinylchloride, and polyethylene. Hydrophilic polymers include, but are notlimited to, cellulosic polymers such as methyl and ethyl cellulose,hydroxyalkylcelluloses such as hydroxypropyl-cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, andCarbopol® 934, polyethylene oxides and mixtures thereof. Fatty compoundsinclude, but are not limited to, various waxes such as carnauba wax andglyceryl tristearate and wax-type substances including hydrogenatedcastor oil or hydrogenated vegetable oil, or mixtures thereof.

In certain preferred forms, the plastic material is a pharmaceuticallyacceptable acrylic polymer, including but not limited to, acrylic acidand methacrylic acid copolymers, methyl methacrylate, methylmethacrylate copolymers, ethoxyethyl methacrylates, cyanoethylmethacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid),poly(methacrylic acid), methacrylic acid alkylamine copolymerpoly(methyl methacrylate), poly(methacrylic acid)(anhydride),polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), andglycidyl methacrylate copolymers.

In certain preferred forms, the acrylic polymer is comprised of one ormore ammonio methacrylate copolymers. Ammonio methacrylate copolymersare well known in the art, and are described in NF XVII as fullypolymerized copolymers of acrylic and methacrylic acid esters with a lowcontent of quaternary ammonium groups.

In one preferred form, the acrylic polymer is an acrylic resin lacquersuch as that which is commercially available from Rohm Pharma under thetradename EUDRAGIT®. In further preferred forms, the acrylic polymercomprises a mixture of two acrylic resin lacquers commercially availablefrom Rohm Pharma under the tradenames EUDRAGIT® RL30D and EUDRAGIT®RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT® RS30D are copolymersof acrylic and methacrylic esters with a low content of quaternaryammonium groups, the molar ratio of ammonium groups to the remainingneutral (meth)acrylic esters being 1:20 in EUDRAGIT® RL30D and 1:40 inEUDRAGIT® RS30D. The mean molecular weight is about 150,000. EUDRAGIT®S-100 and EUDRAGIT® L-100 are also preferred. The code designations RL(high permeability) and RS (low permeability) refer to the permeabilityproperties of these agents. EUDRAGIT® RL/RS mixtures are insoluble inwater and in digestive fluids. However, multiparticulate systems formedto include the same are swellable and permeable in aqueous solutions anddigestive fluids.

The polymers described above such as EUDRAGIT® RL/RS may be mixedtogether in any desired ratio in order to ultimately obtain asustained-release formulation having a desirable dissolution profile.Desirable sustained-release multiparticulate systems may be obtained,for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGITt® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. One skilled in the artwill recognize that other acrylic polymers may also be used, such as,for example, EUDRAGIT® L.

Alternatively, extended release formulations can be prepared usingosmotic systems or by applying a semi-permeable coating to the dosageform. In the latter case, the desired drug release profile can beachieved by combining low permeable and high permeable coating materialsin suitable proportion.

The devices with different drug release mechanisms described above canbe combined in a final dosage form comprising single or multiple units.Examples of multiple units include, but are not limited to, multilayertablets and capsules containing tablets, beads, or granules An immediaterelease portion can be added to the extended release system by means ofeither applying an immediate release layer on top of the extendedrelease core using a coating or compression process or in a multipleunit system such as a capsule containing extended and immediate releasebeads.

Extended release tablets containing hydrophilic polymers are prepared bytechniques commonly known in the art such as direct compression, wetgranulation, or dry granulation. Their formulations usually incorporatepolymers, diluents, binders, and lubricants as well as the activepharmaceutical ingredient. The usual diluents include inert powderedsubstances such as starches, powdered cellulose, especially crystallineand microcrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders. Typical diluentsinclude, for example, various types of starch, lactose, mannitol,kaolin, calcium phosphate or sulfate, inorganic salts such as sodiumchloride and powdered sugar. Powdered cellulose derivatives are alsouseful. Typical tablet binders include substances such as starch,gelatin and sugars such as lactose, fructose, and glucose. Natural andsynthetic gums, including acacia, alginates, methylcellulose, andpolyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilicpolymers, ethylcellulose and waxes can also serve as binders. Alubricant is necessary in a tablet formulation to prevent the tablet andpunches from sticking in the die. The lubricant is chosen from suchslippery solids as talc, magnesium and calcium stearate, stearic acidand hydrogenated vegetable oils.

Extended release tablets containing wax materials are generally preparedusing methods known in the art such as a direct blend method, acongealing method, and an aqueous dispersion method. In the congealingmethod, the drug is mixed with a wax material and either spray-congealedor congealed and screened and processed.

(2) Delayed Release Dosage Forms

Delayed release formulations can be created by coating a solid dosageform with a polymer film, which is insoluble in the acidic environmentof the stomach, and soluble in the neutral environment of the smallintestine.

The delayed release dosage units can be prepared, for example, bycoating a drug or a drug-containing composition with a selected coatingmaterial. The drug-containing composition may be, e.g., a tablet forincorporation into a capsule, a tablet for use as an inner core in a“coated core” dosage form, or a plurality of drug-containing beads,particles or granules, for incorporation into either a tablet orcapsule. Preferred coating materials include bioerodible, graduallyhydrolyzable, gradually water-soluble, and/or enzymatically degradablepolymers, and may be conventional “enteric” polymers. Enteric polymers,as will be appreciated by those skilled in the art, become soluble inthe higher pH environment of the lower gastrointestinal tract or slowlyerode as the dosage form passes through the gastrointestinal tract,while enzymatically degradable polymers are degraded by bacterialenzymes present in the lower gastrointestinal tract, particularly in thecolon. Suitable coating materials for effecting delayed release include,but are not limited to, cellulosic polymers such as hydroxypropylcellulose, hydroxyethyl cellulose, hydroxymethyl cellulose,hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, hydroxypropylmethyl cellulose phthalate, methylcellulose,ethyl cellulose, cellulose acetate, cellulose acetate phthalate,cellulose acetate trimellitate and carboxymethylcellulose sodium;acrylic acid polymers and copolymers, preferably formed from acrylicacid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate, and other methacrylic resinsthat are commercially available under the tradename Eudragit® (RohmPharma; Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55(soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 andabove), EUDRAGIT® S (soluble at pH 7.0 and above, as a result of ahigher degree of esterification), and EUDRAGITS® NE, RL and RS(water-insoluble polymers having different degrees of permeability andexpandability); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymer;enzymatically degradable polymers such as azo polymers, pectin,chitosan, amylose and guar gum; zein and shellac. Combinations ofdifferent coating materials may also be used. Multi-layer coatings usingdifferent polymers may also be applied.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

The coating composition may include conventional additives, such asplasticizers, pigments, colorants, stabilizing agents, glidants, etc. Aplasticizer is normally present to reduce the fragility of the coating,and will generally represent about 10 wt. % to 50 wt. % relative to thedry weight of the polymer. Examples of typical plasticizers includepolyethylene glycol, propylene glycol, triacetin, dimethyl phthalate,diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethylcitrate, tributyl citrate, triethyl acetyl citrate, castor oil andacetylated monoglycerides. A stabilizing agent is preferably used tostabilize particles in the dispersion. Typical stabilizing agents arenonionic emulsifiers such as sorbitan esters, polysorbates andpolyvinylpyrrolidone. Glidants are recommended to reduce stickingeffects during film formation and drying, and will generally representapproximately 25 wt. % to 100 wt. % of the polymer weight in the coatingsolution. One effective glidant is talc. Other glidants such asmagnesium stearate and glycerol monostearates may also be used. Pigmentssuch as titanium dioxide may also be used. Small quantities of ananti-foaming agent, such as a silicone (e.g., simethicone), may also beadded to the coating composition.

III. Methods of Making and Using

Methods of making and using compounds disclosed herein are alsodisclosed.

A. Synthetic Methods

The compounds disclosed herein can be readily synthesized following thegeneric schemes outlined below and specific synthesis methods asdisclosed in the Examples. In one aspect, a generic scheme forsynthesizing the disclosed compounds is General Synthetic Scheme I.

A round bottom flask is charged with ethyl 4-hydroxy-3-methoxybenzoate(4.95 g, 1.0 eq) and acetonitrile (100 mL). To this solution is thensequentially added NEt₃ (5.27 mL, 1.5 eq) and chloromethyl methylether(2.01 mL, 1.05 eq) and the resulting solution is stirred at 50° C.Additional Et₃N and MOMCl are added as necessary to drive the reactionto completion. Upon completion, the solution is cooled to roomtemperature and partially concentrated. The solution is then dilutedwith EtOAc and sequentially washed with ammonium chloride (aq.), water,and brine. The organic portion is dried over sodium sulfate andconcentrated in vacuo to give ethyl 3-methoxy-4-(methoxymethoxy)benzoate(6.06 g) as a pale yellow oil which is used without furtherpurification. ¹H NMR (500 MHz, Chloroform-d) δ 7.57 (dd, J=8.5, 2.0 Hz,1H), 7.51 (d, J=2.0 Hz, 1H), 7.09 (d, J=8.4 Hz, 1H), 5.21 (s, 2H), 4.29(q, J=7.2 Hz, 2H), 3.86 (s, 3H), 3.43 (s, 3H), 1.31 (t, J=7.2 Hz, 3H).

A round bottom flask is charged with ethyl3-methoxy-4-(methoxymethoxy)benzoate (6.06 g, 1.0 eq), THE (30 mL) andwater (15 mL). To this solution is then added LiOH (2.4 g, 4.0 eq) andthe resulting mixture is warmed to 65° C. Upon completion of thereaction (by LCMS), the solution is cooled to room temperature, treatedwith a 10% citric acid solution and thrice extracted with EtOAc. Thecombined organic portions are washed with brine and dried over sodiumsulfate. Concentration in vacuo gives3-methoxy-4-(methoxymethoxy)benzoic acid (3.99 g, 75% yield) as a whitesolid which is clean by NMR. mp=158-160° C. ¹H NMR (500 MHz,Chloroform-d) δ 7.76 (dd, J=8.5, 1.9 Hz, 1H), 7.66 (d, J=2.0 Hz, 1H),7.23 (d, J=8.4 Hz, 1H), 5.34 (s, 2H), 3.98 (s, 3H), 3.55 (s, 3H).

HATU (3.0 eq) is weighed into a vial and dry acetonitrile (0.2 M) isadded. A carboxylic acid (1.05 eq) is added followed by Hünig's base(3.0 eq). The mixture is stirred at 23° C. for 5 min before the additionof methyl 3-amino-4-hydroxybenzoate (1.0 eq). After stirring for 18 h at23° C., the reaction is diluted with dichloromethane and extracted with1N HCl, sat. NaHCO₃ solution (2×) and brine. The organic layer is driedover anhydrous Na₂SO₄ and evaporated. The residue is chromatographed bysilica gel flash chromatography and elution with 0-20% ethyl acetate inhexanes to give the expected N-acylated product.

A flask is charged with an acetophenone (1.0 eq) and THE (1.0 M, 2 mL).The solution is then diluted with chloroform (0.33 M, 6 mL) and AcOH(0.065 mL). Br₂ (1.1 eq) is then slowly added to the solution (an icebath was used for larger scale reactions). The solution is stirred untilthe characteristic bromine color faded (˜30 min). To the solution isthen added thiourea (1.5 eq) and the resulting mixture stirred overnight(˜18 h). The resulting mixture contains a precipitate which is collectedvia filtration, washing sequentially with dichloromethane and water. Thesolid is dissolved in THE and concentrated in vacuo to aid in drying.The resulting 2-aminothiazole is used without further purification.

A microwave vial is charged with an α-bromoacetophenone (1.0 eq) and 1:1EtOH/water (0.5 M). Thiourea (1.1 eq) is added, and the mixture warmedin the microwave reactor at 75° C. for 30 min. Upon completion, the roomtemperature solution contains a precipitate which could be collected viafiltration (washing with water), and dried to give the desired2-aminothiazole.

A vial is charged with the 2-aminothiazole (1.0 eq), triethylamine (3.0eq), and acetonitrile (˜0.4 M). To this solution is added theacetal-protected carboxylic acid (1.0 eq). Finally, HATU (1.0 eq) isadded and the vial is warmed to 70° C. Stirring at 70° C. is continueduntil full consumption of the carboxylic acid by LCMS. The solution iscooled to room temperature, diluted with EtOAc, and thrice washed withwater. Concentration in vacuo gives an oil that was is without furtherpurification. This oil is directly dissolved in THE (4 mL) and treatedwith 1N HCl (1 mL). The solution is warmed to 65° C. and stirred untilthe acetal deprotection is complete by LCMS. Upon completion, thesolution is cooled to room temperature, diluted with EtOAc andsequentially washed with water and brine. Concentration and purificationon silica gel (hex/EtOAc gradient 5-50%) gives the clean phenol as awhite solid.

A vial is charged with the appropriate phenol (1.0 eq) and DMF (0.1 M).Potassium carbonate (3.0 eq) is added, followed by an α-chloroacetamide(1.1 eq). This solution is warmed to 70° C. and stirred until completeby LCMS (˜2-4 h). Upon completion, the solution is cooled to roomtemperature and diluted with water. In most instances, the addition ofwater initiated the precipitation of the product from the solution,which could then be collected via filtration (washing with water). Ifprecipitation does not occur, then the reaction is diluted with EtOAc,thrice washed with water, and concentrated. Purification of either theconcentrated material, or the precipitated solid is performed either onsilica gel (hex/EtOAc gradient 50%-100%) or mass-triggered, reversephase preparative HPLC (water/MeCN gradient).

A vial is charged with an appropriate phenol (1.0 eq) and potassiumcarbonate (2.0 eq) in acetonitrile (0.2 M). To this mixture is added anα-bromoacetate (1.05 eq), which is warmed to 75° C. Upon completion (˜1h), the solution is decanted from the remaining carbonate salts and thenpurified directly on silica gel (hex/EtOAc gradient 5%-60%) to give theintermediate ester as a tan solid. This material (1.0 eq) is added to asolution of THE (3 mL) and water (1.5 mL). LiOH is then added (4 eq) andstirred at room temperature until the saponification is complete (1.5h). The solution is quenched with 1N HCl and twice extracted with EtOAc.The combined organic portions are dried over sodium sulfate andconcentrated in vacuo to give the expected carboxylic acid as a whitesolid. This carboxylic acid (1.0 eq) is dissolved in DMF (0.1 M) andtreated with triethylamine (2.0 eq) and various amines (1.2 eq). Lastly,HATU (1.2 eq) is added and stirred for 60 min. At this time the reactionis complete and the solution is purified directly on a reverse phasepreparatory HPLC to give the corresponding amides as white solids.

In another aspect, a generic scheme for synthesizing the disclosedcompounds is General Synthetic Scheme 2.

The commercially available 4-((2-ethoxy-2-oxoethyl)amino)benzoic acid(1.0 eq) is dissolved in acetonitrile (0.3 M) and treated withtriethylamine (4.0 eq) and a 2-aminothiazole (1.0 eq). To this mixtureis added HATU (1.5 eq). The resulting solution is warmed to 75° C. andstirred until the reaction was complete. At this time the solution iscooled to room temperature and diluted with EtOAc. The solution issequentially washed with water (2×) and brine, dried over sodiumsulfate, and then concentrated in vacuo. Purification on silica gel(hex/EtOAc gradient 10%-100%) gives an impure mixture which could bedissolved in DMF and precipitated by the addition of water. This solidis dissolved in THF:H₂O (3:1) and treated with LiOH (4.0 eq).Saponification is completed within one hour of stirring at roomtemperature. The solution is then diluted with EtOAc and washed with a10% citric acid solution followed by brine. Drying on sodium sulfate andconcentration gives a colored solid which is then washed with adichloromethane and methanol solution to give the intermediatecarboxylic acid as an off-white solid. This carboxylic acid (1.0 eq) isdissolved in DMF (0.25 M). Triethylamine (2.0 eq) and an amine (4.0 eq)are then added. Lastly, HATU (1.2 eq) is added and the solution isstirred at room temperature until the reaction is complete. Water isthen slowly added to the reaction which then formed a precipitate. Thisprecipitate was collected via filtration and washed with a mixture ofTHE and methanol to cleanly the expected amide. If precipitation did notoccur, then the mixture could be purified via reverse phase HPLC to givethe expected product.

In another aspect, a generic scheme for synthesizing the disclosedcompounds is General Synthetic Scheme 3.

The commercially available 3-methoxy-4-nitrobenzoic acid (1.0 eq) isdissolved in acetonitrile (0.25 M) and treated with triethylamine (2.0eq) and HATU (1.0 eq). To this mixture is added4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq). The resulting solution iswarmed to 65° C. and stirred until the reaction is complete. At thistime the solution is cooled to room temperature and diluted with water.The resulting precipitate was collected via filtration, washed withadditional water, and dried in vacuo to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide as a paleyellow solid (82% yield). A dry round bottom flask is charged with 10%palladium on carbon (0.05 eq) under an atmosphere of nitrogen. EtOAc(˜0.2 M) is then added, followed by3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide (1.0 eq).A hydrogen-filled balloon is then affixed to the flask and the airspacewas evacuated and back-filled with hydrogen. The mixture is vigorouslystirred until the reaction was complete, adding additional catalyst asneeded and some methanol to ensure solubility. The mixture is thenfiltered through celite, washing with methanol to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide as ayellow solid. This solid (1.0 eq) is then dissolved in acetonitrile (0.1M) and treated with excess glyoxylic acid (˜15 eq of a 50% aqueoussolution). Sodium cyanoborohydride (5.0 eq) is added and stirred at roomtemperature until the reaction was complete. The solution is quenchedwith acetic acid, and partitioned between EtOAc and 1N HCl. The organicportion is concentrated to a yellow solid that is used without furtherpurification. The crude(2-methoxy-4-((4-(4-methoxyphenyl)thiazol-2-yl)carbamoyl) phenyl)glycine(1.0 eq) is dissolved in DMF and treated with morpholine (10.0 eq). Tothis room temperature solution is added HATU (3.0 eq) and stirring iscontinued until the reaction is complete. The solution is then quenchedwith water which initiates the formation of a white precipitate. Thisimpure solid is collected via filtration and then purified on silica gel(hexane/EtOAc gradient 35%-100%) to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-((2-morpholino-2-oxoethyl)amino)benzamideas a white solid. Other compounds can be prepared using this genericscheme as exemplified herein.

In still another aspect, a generic scheme for synthesizing the disclosedcompounds is General Synthetic Scheme 4.

A vial is charged with the 5-bromothiazol-2-amine hydrobromide (1.0 eq),triethylamine (3.0 eq), and DMF (˜0.2 M). To this solution is added the3-methoxy-4-(methoxymethoxy)benzoic acid (1.2 eq). Finally, HATU (1.2eq) is added and the vial was warmed to 75° C. Stirring at 75° C. iscontinued until full consumption of the carboxylic acid by LCMS. Thesolution is then cooled to room temperature, diluted with EtOAc, andwashed with brine. The solution is concentrated in vacuo and was usedwithout further purification. This material is dissolved in THE (4 mL)and treated with 1N HCl (1 mL). The solution is then warmed to 65° C.and stirred until the acetal deprotection was complete by LCMS. Uponcompletion, the solution was cooled to room temperature, diluted withEtOAc and sequentially washed with NaHCO₃ and brine. The solution isconcentrated in vacuo, and purified on silica gel (hex/EtOAc gradient10-75%) to give the clean phenol as a white solid.

This phenol (1.0 eq) is transferred to a microwave vial and treatedsequentially with 1,4-dioxane/water (4:1), an aryl boronic acid (1.5 eq)and sodium carbonate (2.0 eq). The vial is flushed with argon and thencharged with PdCl₂(dppf) (0.1 eq). The mixture is briefly sparged withargon and then heated in a sealed microwave vial at 100° C. for 60 min.The solution is poured into water and twice extracted with EtOAc. Theorganic portion is dried over sodium sulfate, concentrated in vacuo, andpurified on silica gel (hexane/EtOAc gradient 10-60%) to give theexpected Suzuki product.

This phenol (1.0 eq) is dissolved in acetonitrile and treated withpotassium carbonate (2.0 eq). Lastly, 2-chloro-1-morpholinoethan-1-one(1.0 eq) is added and this solution warmed to 85° C. and stirred for 4h. The solution is cooled to room temperature and the inorganic saltsare removed via filtration. The solution is then purified either onreverse phase preparatory HPLC, or silica gel to give the expectedproduct as a white solid.

In still another aspect, a generic scheme for synthesizing the disclosedcompounds is General Synthetic Scheme 5.

In still another aspect, a generic scheme for synthesizing the disclosedcompounds is General Synthetic Scheme 6.

B. Administration

The compounds described herein can be administered in an effectiveamount to a subject that is in need of alleviation or amelioration fromone or more symptoms associated with cellular lipotoxicity or insulinresistance. Additionally, the data disclosed herein shows that SBI-993treatment resulted in a small but significant reduction in body weightas compared to vehicle. Accordingly, method for reducing body weight ina subject in need thereof, are also contemplated and disclosed.

As will be pointed out below, the exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the disease that is being treated, theparticular compound used, its mode of administration, and the like.Thus, it is not possible to specify an exact “effective amount.”However, an appropriate effective amount can be determined by one ofordinary skill in the art using only routine experimentation. Thedosages or amounts of the compounds described herein are large enough toproduce the desired effect in the method by which delivery occurs. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the subject and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician based onthe clinical condition of the subject involved. The dose, schedule ofdoses and route of administration can be varied.

The compositions are administered in an effective amount and for aperiod of time effect to reduce one or more symptoms associated with thedisease to be treated. Exemplary symptoms include, but are not limitedto higher than normal blood glucose levels or higher than normalcellular lipid/TAG levels. It is within the abilities of one of ordinaryskill in the art to determine higher than normal blood glucose cellularlipid/TAG levels, which are used herein to refer to levels found in ahealthy individual.

In preferred forms, patients to be treated include those with noninsulindependent diabetes mellitus (NIDDM or Type II diabetes), insulindependent diabetes mellitus (IDDM or Type I diabetes), insulinresistance such as impaired glucose tolerance. Insulin resistance isdefined as a state in which circulating insulin levels in excess of thenormal response to a glucose load are required to maintain theeuglycemic state (Ford, et al. JAMA (2002) 287:356-9). Insulinresistance, and the response of a subject with insulin resistance totherapy, may be quantified by assessing the homeostasis model assessmentto insulin resistance (HOMA-IR) score, a reliable indicator of insulinresistance (Katsuki, et al. Diabetes Care 2001; 24:362-5,). The estimateof insulin resistance by the homeostasis assessment model (HOMA)-IRscore is calculated with the formula (Galvin, et al. Diabet Med 1992;9:921-8): HOMA-IR=[fasting serum insulin (μU/mL)]×[fasting plasmaglucose (mmol/L)/22.5] Subjects with a predisposition for thedevelopment of impaired glucose tolerance (IT) or type 2 diabetes arethose having euglycemia with hyperinsulinenia are by definition, insulinresistant. A typical subject with insulin resistance is usuallyoverweight or obese.

However, the compositions disclosed herein can be administered tosubjects with any disorder associated with cellular lipotoxicity,abnormally increased TAG synthesis and/or deposition, etc. or subjectswho can benefit from increasing insulin sensitivity and/or increasedcellular glucose uptake. Examples include subjects with obesity,including insulin resistant obesity, obesity or diabetes-related heartdisease (including atheromatous disease), metabolic syndrome,non-alcoholic fatty liver disease including hepatic steatosis andnonalcoholic steatohepatitis, triglyceride storage disease, dysfunctionsassociated with lipid biosynthesis and triglyceride levels, as seen forexample in renal lipotoxicity-associated inflammation, diabeticnephropathy, pancreatic beta cell lipotoxicity-induced dysfunction(Diabetes, 50(Suppl 1:S118-21 (2001).

The compounds disclosed herein are potent inhibitors of MondoA. Carroll,et al. identified a critical role for lipid biosynthesis in survival ofMyc-driven cancer requiring MondoA in different cancer types includingneuroblastoma, lung squamous cell carcinoma/lung adenocarcinoma, liverhepatocellular carcinoma, colon adenocarcinoma, acute myeloid leukemia,and breast invasive carcinoma. Carroll, et al., Cancer Cell 2015;27(2):271-285). Their studies showed that knockdown of MondoAsignificantly reduces survival of human B cells expressing c-Myc.Accordingly, methods for reducing MondoA activity/function within thecontext of Mvc-driven cancers are also contemplated and disclosedherein.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Materials and Methods Cell Culture

Primary human skeletal myoblasts, kindly provided by Dr. Steven Smith(Translational Research Institute for Metabolism and Diabetes, FloridaHospital, Orlando, Fla.), were isolated and cultured from lean healthymale subjects obtained by muscle biopsy. Myoblasts were grown toapproximately 80-90% confluence and differentiated into myotubes aspreviously described (Sparks, et al., PLoS One, 2011; 6(7):e21068). Ratheart myoblast H9c2 cells (ATCC) were maintained in Dulbecco's modifiedEagle medium (DMEM) containing 10% fetal bovine serum (FBS) in a 5% CO2incubator. At approximately 80-90% confluence, H9c2 myoblasts weredifferentiated into myotubes by culturing in DMEM supplemented with 1%FBS for 4-5 days.

Fatty Acid Oxidation (FAO) Assay

FAO rates were determined for primary human skeletal myotubes with orwithout SBI-477 using 125 μM [3H]-palmitic acid (Djouadi, et al., MolGenet Metab. 2003; 78(2):112-118). Primary human skeletal myotubes weregrown and differentiated in 24-well plates. Cells were treated with theindicated concentration of SBI-477 for 24 hours. Following compoundtreatment, cells were rinsed three times with PBS and then incubated in125 μM [3H]-palmitic acid (60 Ci/mmol) bound to fatty acid free albumincontaining 1 mM carnitine for 2 hours at 37° C. The cell medium wastransferred to a tube containing cold 10% trichloroacetic acid (TCA).The tubes were centrifuged at 8,500×g for 10 minutes at 4° C. Thesupernatant was immediately removed, mixed with 6N NaOH, and applied toion-exchange resin (DOWEX 1; Sigma-Aldrich). The eluate was collected,measured by liquid scintillation analyzer (PerkinElmer) and normalizedto total protein amount. The amount of cell protein was measured byMicro BCA protein assay kit (Thermo Scientific).

SBI-477 and SBI-993 Synthesis

Synthesis Route for SBI-477

A round bottom flask was charged with ethyl 4-hydroxy-3-methoxybenzoate(4.95 g, 1.0 eq) and acetonitrile (100 mL). To this solution was thensequentially added NEt3 (5.27 mL, 1.5 eq) and chloromethyl methylether(2.01 mL, 1.05 eq); the resulting solution was stirred at 50° C.Additional Et3N and MOMCl were added as necessary to drive the reactionto completion. Upon completion, the solution was cooled to roomtemperature and partially concentrated. The solution was then dilutedwith EtOAc and sequentially washed with aqueous ammonium chloride,water, and brine. The organic portion was dried over sodium sulfate andconcentrated in vacuo to give ethyl 3-methoxy-4-(methoxymethoxy)benzoate(6.06 g) as a pale yellow oil which was used without furtherpurification. 1H NMR (500 MHz, Chloroform-d) δ 7.57 (dd, J=8.5, 2.0 Hz,1H), 7.51 (d, J=2.0 Hz, 1H), 7.09 (d, J=8.4 Hz, 1H), 5.21 (s, 2H), 4.29(q, J=7.2 Hz, 2H), 3.86 (s, 3H), 3.43 (s, 3H), 1.31 (t, J=7.2 Hz, 3H).

A round bottom flask was charged with ethyl3-methoxy-4-(methoxymethoxy)benzoate (6.06 g, 1.0 eq), THE (30 mL) andwater (15 mL). To this solution was then added LiOH (2.4 g, 4.0 eq) andthe resulting mixture was warmed to 65° C. Upon completion of thereaction (by LCMS), the solution was cooled to room temperature, treatedwith a 10% citric acid solution and thrice extracted with EtOAc. Thecombined organic portions were washed with brine and dried over sodiumsulfate. Concentration in vacuo gave 3-methoxy-4-(methoxymethoxy)benzoicacid (3.99 g, 75% yield) as a white solid which was pure by NMR.mp=158-160° C. 1H NMR (500 MHz, Chloroform-d) δ 7.76 (dd, J=8.5, 1.9 Hz,1H), 7.66 (d, J=2.0 Hz, 1H), 7.23 (d, J=8.4 Hz, 1H), 5.34 (s, 2H), 3.98(s, 3H), 3.55 (s, 3H).

A vial was charged with 4-(4-methoxyphenyl)thiazol-2-amine (412 mg, 1.0eq), triethylamine (558 μL, 2.0 eq), and acetonitrile (˜0.4 M). To thissolution was then added 3-methoxy-4-(methoxymethoxy)benzoic acid (509mg, 1.2 eq). Finally, HATU (912 mg, 1.0 eq) was added and the vial waswarmed to 75° C. Stirring at 75° C. was continued until full consumptionof the carboxylic acid by LCMS. The solution was then cooled to roomtemperature, diluted with EtOAc, and then sequentially washed with waterand brine. Concentration in vacuo gave an oil that was used withoutfurther purification. This oil was directly dissolved in THE (16 mL) andtreated with 1N HCl (4 mL). The solution was then warmed to 65° C. andstirred until the acetal deprotection was complete by LCMS (45 min).Upon completion, the solution was cooled to room temperature, dilutedwith EtOAc and sequentially washed with saturated NaHCO3, water, andbrine. After drying over sodium sulfate, the material was concentratedand purified on silica gel (10-50% hex/EtOAc gradient) to give4-hydroxy-3-methoxy-N-(4-(4-ethoxyphenyl)thiazol-2-yl)benzamide as awhite solid (441 mg, 62% yield). 1H NMR (500 MHz, Chloroform-d) δ 7.76(d, J=8.8 Hz, 2H), 7.57 (d, J=2.0 Hz, 1H), 7.45 (dd, J=8.3, 2.1 Hz, 1H),7.04 (s, 1H), 7.01 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.8 Hz, 2H), 3.99 (s,3H), 3.85 (s, 3H).

A vial was charged with4-hydroxy-3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamide (441mg, 1.0 eq) and DMF (0.1 M). Potassium carbonate (343 mg, 2.0 eq) wasadded, followed by 2-chloro-1-morpholinoethan-1-one (163 μL, 1.0 eq).This solution was then warmed to 85° C. in a microwave reactor for 4hours. The solution was partially concentrated and then partitionedbetween EtOAc and water. The aqueous layer was further extracted withEtOAc and the combined organic layers were dried over sodium sulfate.Purification on silica gel (10-100% hex/EtOAc gradient) gave3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholino-2-xoethoxy)benzamide(398 mg, 66%) as a white solid. 1H NMR (500 MHz, methanol-d4) δ 7.87 (d,J=8.7 Hz, 2H), 7.69 (s, 1H), 7.69-7.62 (m, 1H), 7.26 (s, 1H), 7.08 (d,J=8.4 Hz, 1H), 6.96 (d, J=8.8 Hz, 2H), 4.94 (s, 2H), 3.97 (s, 3H), 3.83(s, 3H), 3.69 (m, 4H), 3.61 (m, 4H). 13C NMR (125 MHz, methanol-d4) δ166.10, 164.42, 159.76, 158.80, 151.04, 150.15, 149.73, 127.58, 127.38,126.00, 120.52, 114.28, 112.98, 111.43, 106.61, 68.53, 67.02, 66.95,56.21, 55.53, 46.18, 42.80.

Synthesis Route for SBI-993

The commercially available 4-((2-ethoxy-2-oxoethyl)amino)benzoic acid(1.21 g, 1.0 eq) was dissolved in acetonitrile (0.3 M) and treated withtriethylamine (3.0 mL, 4.0 eq) and a 4-(4-methoxyphenyl)thiazol-2-aminehydrobromide (1.56 g, 1.0 eq). To this mixture was then added HATU (3.09g, 1.5 eq). The resulting solution was warmed to 75° C. and stirreduntil the reaction was complete (˜ 48 h). At this time the solution wascooled to room temperature and diluted with EtOAc. The solution was thensequentially washed with water (2×) and brine, dried over sodiumsulfate, and then concentrated in vacuo. Purification on silica gel(hex/EtOAc gradient 10%-100%) gave an impure mixture which could bedissolved in DMF and precipitated by the addition of water. This solidwas dissolved in THE (12 mL) and water (4 mL) and treated with LiOH (308mg, 4.0 eq). Saponification was completed within one hour of stirring atroom temperature. The solution was then diluted with EtOAc and washedwith a 10% citric acid solution followed by brine. Drying on sodiumsulfate and concentration gave a colored solid, which was then washedwith a dichloromethane and methanol solution to give the intermediatecarboxylic acid as an off-white solid (571 mg). This carboxylic acid(570 mg, 1.0 eq) was dissolved in DMF (0.25 M). Triethylamine (415 μL,2.0 eq) and morpholine (518 μL, 4.0 eq) were then added. Lastly, HATU(680 mg, 1.2 eq) was added and the solution was stirred at roomtemperature for 2 h. Water was then slowly added to the reaction whichthen formed a precipitate. This precipitate was collected via filtrationand washed with a mixture of THF and methanol to cleanly giveN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-((2-morpholino-2-oxoethyl)amino)benzamide(422 mg) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 12.22 (s, 1H),7.95 (d, J=8.7 Hz, 2H), 7.88 (d, J=8.8 Hz, 2H), 7.44 (s, 1H), 7.01 (d,J=8.8 Hz, 2H), 6.76 (d, J=8.8 Hz, 2H), 6.48 (t, J=5.2 Hz, 1H), 4.05 (d,J=5.1 Hz, 2H), 3.80 (s, 3H), 3.61 (m, 4H), 3.56-3.47 (m, 4H).

Microarray Studies

Differentiated primary human myotubes were incubated with 10 μM SBI-477,1 M A922500 (DGAT inhibitor) or DMSO vehicle control for 24 hours. TotalRNA was extracted using RNABee (Amsbio, Lake Forest, Calif.) and cleanedup using the RNeasy column (Qiagen, Valencia, Calif.) followingmanufacturer protocols. The Genomics core at Sanford Burnham Prebys-LakeNona performed the hybridization to a GeneChip® Human gene 1.0 ST Array(Affymetrix, Santa Clara, Calif.). Two independent samples were analyzedfor each group. Affymetrix experiment console version 1.1 was employedto normalize array data using the RMA approach. R package LIMMA (LinearModel for Microarray Data) was applied for differential gene expressionanalysis. The gene array data discussed in this publication have beendeposited in NCBI's Gene Expression Omnibus and are accessible throughGEO Series accession number GSE77212.

Lipidomics

Quantitative lipidomic analyses was performed on human skeletal myotubesexposed to bovine albumin serum (BSA) or 100 μM oleate in the presenceof DMSO vehicle (white bars) or 10 μM SBI-477 (black bars) for 24 hours.

Primary human myotubes were treated with BSA or 100 μM oleate with 10 μMSBI-477 or a vehicle control for 24 hours. The cells were added to 300μL PBS in an Eppendorf tube and homogenized for 1 minute using adisposable soft tissue homogenizer. A 25 μL aliquot was used todetermine the protein content (BCA protein assay kit, Thermo Scientific,Rockford, Ill.). The remaining homogenate was accurately transferredinto a disposable glass culture test tube, and a mixture of lipidinternal standards was added prior to lipid extraction forquantification of all reported lipid species. Lipid extraction wasperformed by using a modified Bligh and Dyer procedure as describedpreviously (Wang and Han, 2014). Each lipid extract was resuspended intoa volume of 500 μL of chloroform/methanol (1:1, v/v) per mg of proteinand flushed with nitrogen, capped, and stored at −20° C. for lipidanalysis. For ESI direct infusion analysis, lipid extract was furtherdiluted to a final concentration of ˜500 fmol/μL, and the massspectrometric analysis was performed on a QqQ mass spectrometer (ThermoTSQ VANTAGE, San Jose, Calif.) equipped with an automated nanospraydevice (TriVersa NanoMate, Advion Bioscience Ltd., Ithaca, N.Y.).

Glycogen Synthesis Assay

Human skeletal myotubes were incubated with or without SBI-477 and thentreated with or without insulin (100 nM) for 30 minutes. Glycogensynthesis rates were then measured as described (Halse, et al., J BiolChem. 1999; 274(2):776-780). Briefly, differentiated primary humanskeletal myotubes were treated with SBI-477 as described in the glucoseuptake assay. Following incubation with SBI-477, cells were serumstarved in α-MIEM for 1 hour. The medium was replaced with α-MIEMsupplemented with [14C]-D-glucose (1 μCi/mL) for three hours and thentreated in the absence or presence of insulin (100 nM) for 30 minutes.After incubation, the cells were washed three times with ice-cold PBSand then lysed in 30% potassium hydroxide (KOH) containing 6 mg/mLglycogen. For glycogen precipitation, the samples were added to ice-cold100% ethanol for 18 hours at 4° C. The samples were centrifuged at8,500×g for 10 minutes at 4° C. and the supernatant was discarded. Afterone wash with 75% ethanol, the glycogen precipitate was dissolved indistilled water. [14C]-D-glucose incorporated to glycogen was counted byliquid scintillation analyzer (PerkinElmer) and normalized to totalprotein.

Glucose Tolerance Test and Insulin Signaling

Following a 5 hour fast, a blood sample was obtained from the tail tipfor the measurement of baseline glucose using a hand-held glucometer(Accu-Chek Aviva Plus, Roche). A bolus of glucose (1 g/kg) was thenadministered via i.p. injection. Blood samples were obtained at 5, 15,30, 60 and 90 minutes following the glucose bolus for measurement ofblood glucose. For detection of insulin signaling, mice were fasted for5 hours on the last day of the study. Gastrocnemius skeletal muscle andliver tissues were harvested 10 minutes after an acute insulinadministration (1.5 U/kg, i.p.) and immediately snap frozen. The tissuesamples were homogenized in RIPA buffer containing 1% NP-40, 5 mMNa4P2O7, 1 mM EDTA, 20 mM NaF, 2 mM Na3VO4, 1× Complete proteaseinhibitor (Roche) and 1 mM phenylmethylsulfonyl fluoride. Western blotanalysis for levels of phospho-Akt (5473) and total Akt was thenperformed.

Myocyte Triglyceride Measurements

Human skeletal myotubes were differentiated for 8 days. On day 7, 100 μMof oleic acid complexed to fatty acid-free BSA was added to the cellswith the indicated concentration of test compound for 24 hours.Following incubation with oleate and compound, cells were formaldehydefixed and stained with AdipoRed™ (Lonza). Triglyceride accumulation wasmeasured using signal intensity at excitation 540/emission 590 nm(Bio-Tek Instruments, Inc.). Dose response curves were generated inPrism 6 (GraphPad Software). EC50 values were calculated usingnon-linear regression analysis. For biochemical measurement oftriglyceride, human skeletal myotubes were incubated with oleate-BSA andtest compound as described above. After 24 hour incubation, cells werecollected with a lysis buffer containing 0.1% IGEPAL CA-630(Sigma-Aldrich) in PBS. The harvested cells were sonicated for 5 secondsand then centrifuged at 10,000×g for 10 minutes at 4° C. The supernatantwas removed and stored on ice. TAG levels were quantified usingInfinity™ Triglycerides Liquid Stable Reagent (Thermo Scientific),following the manufacturer's instructions for colorimetric assay.

Immunoblotting Analysis

Cells were lysed in the RIPA buffer containing 1% NP-40, 5 mM Na4P2O7, 1mM EDTA, 20 mM NaF, 2 mM Na3VO4, 1× cOmplete™ protease inhibitor (Roche)and 1 mM phenylmethylsulfonyl fluoride. Whole cell lysates weresubjected to SDS-PAGE and transferred to a nitrocellulose membrane. Theblots were hybridized with specific antibodies and developed withenhanced chemiluminescence kit (Pierce). The following antibodies wereused: phospho-Akt (Ser473), Akt, phospho-IRS-1 (Ser636/639), S6K,phospho-S6K (T389), AMPKα, phospho-AMPKα (T172), alpha-tubulin, andLamin A/C (Cell Signaling Technology; catalog numbers 4058, 9272, 2388,9202, 9234, 2532, 2531, 3873, and 2032 respectively); β-actin and IRS-1(Y612) (Sigma-Aldrich; catalog numbers A5316 and I2658); IRS-1 and GAPDH(Santa Cruz Biotechnology; catalog numbers SC-560 and SC-25778); TXNIP(MBL; catalog number K0205-3); MondoA (Bethyl Laboratories; catalognumber A303-195A). Protein quantification was performed using FluorChemQ(Alpha Innotech) and normalized to D-actin or total protein whereindicated.

Cellular Glucose Uptake Assay

After differentiation, primary human skeletal myotubes were incubatedwith the indicated concentration of SBI-477 for 24 hours and thentreated with insulin (100 nM) for 30 minutes. Cells were washed inphosphate-buffered saline (PBS) three times at room temperature and thenincubated with Krebs-Ringer HEPES buffer (140 mM NaCl, 5 mM KCl, 1 mMCaCl2, 2.5 mM MgSO4, 2.5 mM, NaH2PO4, 20 mM HEPES and 0.1% bovine serumalbumin) containing [3H]-2-deoxyglucose (1.0 Ci/mL) for 15 min. Glucoseuptake was terminated by washing five times with ice-cold PBS. The cellswere then solubilized with 0.5N sodium hydroxide (NaOH). The amount of[3H]-2-deoxyglucose taken up was measured by liquid scintillationanalyzer (PerkinElmer) and normalized to total protein.

RNA Isolation and Quantitative RT-PCR

Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to themanufacturer's instructions. cDNA was synthesized using the AffinityScript cDNA Synthesis Kit (Stratagene) with 0.5 μg of total RNA. PCRreactions were performed in triplicate The Sanford Burnham PrebysMedical Discovery Institute's Institutional Animal Care and UseCommittee (IACUC) approved all animal studies. Roche LightCycler® 480Instrument II) with specific primers for each gene. Primer sets arelisted in Table 2.

TABLE 2 Primer sequences for qRT-PCR. Gene Forward (5′ to 3′)Reverse (5′ to 3′) Human ACACA (ACC1) GTCAATCTTGAGGGCTAGGTCTCTGGTTCAGCTCCAGAGGTT (SEQ ID NO: 1) (SEQ ID NO: 2) ACACB (ACC2)CAGGTGGGCCTATGAGATGT GGACGTAATGATCCGCCATCTT (SEQ ID NO: 3)(SEQ ID NO: 4) ARRDC4 AGTTTCCTGCATGTTCATTCCT CCACAATTCGGGAACATGTATT(SEQ ID NO: 5) (SEQ ID NO: 6) DGAT1 GTGGCTTCAGCAACTACCGTCAGGAACAGAGAAACCACCTG (SEQ ID NO: 7) (SEQ ID NO: 8) DGAT2GCTCTACTTCACTTGGCTGGT CAGCAGGTTGTGTGTCTTCAC (SEQ ID NO: 9)(SEQ ID NO: 10) ELOVL6 GCAGTCAGTTTGTGACCAGG ATCAGCTTCTGCTTCCTCAGA(SEQ ID NO: 11) (SEQ ID NO: 12) FASN GATGCCTCCTTCTTCGGAGTCCTCGGAGTGAATCTGGGTT (SEQ ID NO: 13) (SEQ ID NO: 14) GPAM(GPAT1)TCAAGAGCGAGATGTGCATAAG CATCAGGGTTTAATTCAGCAG (SEQ ID NO: 15)(SEQ ID NO: 16) MLXIP (MondoA) GCTCACCAAGCTCTTCGAGT GCCGGATCTTGTCTCTCCAC(SEQ ID NO: 17) (SEQ ID NO: 18) MLXIPL (ChREBP) GTGTCTCCCAAGTGGAAGAATTTGCTCTTCCTCCGCTTCACAT (SEQ ID NO: 19) (SEQ ID NO: 20) SCDCTTCTCTCACGTGGGTTGGC ATCAGCAAGCCAGGTTTGTAG (SEQ ID NO: 21)(SEQ ID NO: 22) TXNIP AGTTTCCTGCATGTTCATTCCT CCACAATTCGGGAACATGTATT(SEQ ID NO: 23) (SEQ ID NO: 24) RPLP0 TCTACAACCCTGAAGTGCTTGATATAGAATGGGGTACTGATGCAA (36B4) (SEQ ID NO: 25) (SEQ ID NO: 26) Mouse GeneAcaca (ACC1) GGCCAGTGCTATGCTGAGAT ATCACACAGCCAGGGTCAAG (SEQ ID NO: 27)(SEQ ID NO: 28) Acacb (ACC2) CGCTCACCAACAGTAAGGTGG GCTTGGCAGGGAGTTCCTC(SEQ ID NO: 29) (SEQ ID NO: 30) Dgatl GTGCACAAGTGGTGCATCAGCAGTGGGATCTGAGCCATCA (SEQ ID NO: 31) (SEQ ID NO: 32) Dgat2GCATTTGACTGGAACACGCC CTGGTGGTCAGCAGGTTGTG (SEQ ID NO: 33)(SEQ ID NO: 34) Elov16 CGTAGCGACTCCGAAGATCA AGCGTACAGCGCAGAAAACA(SEQ ID NO: 35) (SEQ ID NO: 36) Fasn CCAAGCAGGCACACACAATGGTTCGTTCCTCGGAGTGAGG (SEQ ID NO: 37) (SEQ ID NO: 38) Gpam (GPAT1)ACAGTTGGCACAATAGACGTTT CTTCCATTTCAGTGTTGCAGA (SEQ ID NO: 39)(SEQ ID NO: 40) G6pc GCTGGAGTCTTGTCAGGCAT GCCGCTCACACCATCTCTTA(SEQ ID NO: 41) (SEQ ID NO: 42) Pklr (LPK) GCTAGGAGCACCAGCATCATTGGGAGAAGTTGAGTCGTGC (SEQ ID NO: 70) (SEQ ID NO: 43) MixCATGGACTCCCTCTTCCAGTC GATGAAGGACACCGATCACA (SEQ ID NO: 44)(SEQ ID NO: 45) Mlxip (MondoA) TGCTACCTGCCACAGGAGTCGACTCAAACAGTGGCTTGATGA (SEQ ID NO: 46) (SEQ ID NO: 47) Mlxipl/(ChREBP)CAGCATCGATCCGACACTCA CGGATCTTGTCCCGGCATAG (SEQ ID NO: 48)(SEQ ID NO: 49) Pepck AGTTCGTGGAAGGCAAT GTGAGAGCCAGCCAACA(SEQ ID NO: 50) (SEQ ID NO: 51) Scdl CCAAGCTGGAGTACGTCTGGCAGAGCGCTGGTCATGTAGT (SEQ ID NO: 52) (SEQ ID NO: 53) TxnipGTCTCAGCAGTGCAAACAGACTT GCTCGAAGCCGAACTTGTACTC (SEQ ID NO: 54)(SEQ ID NO: 55) Rplp0 (36B4) TGGAAGTCCAACTACTTCCTCAAATCTGCTGCATCTGCTTGGAG (SEQ ID NO: 56) (SEQ ID NO: 57)

The expression of Rplp0 (36B4) was used to normalize all gene expressiondata.

Transient Transfection and Luciferase Reporter Assay

Transient transfections were performed using Attractene (Qiagen)according to the manufacturer's protocols. A full length (1.5 kb) andtwo serial deletion (1.0 kb and 0.5 kb) reporters of the human TXNIPpromoter were generously provided by Dr. Fumihiko Urano (WashingtonUniversity School of Medicine). Mutation of the carbohydrate-responsiveelements (ChoREs) of the reporters was generated by QuikChange IISite-Directed Mutagenesis kit (Agilent). H9c2 myoblast cells (ATCC) werecotransfected with 700 ng of the reporter and 25 ng of CMV promoterdriven Renilla luciferase as a control for transfection efficiency.Eighteen hours after transfection, the cells were cultured in DMEMsupplemented with 1% fetal bovine serum to induce differentiation for 4days. SBI-477 was added during the last 24 hours at the indicatedconcentration. After compound incubation, luciferase activity wasmeasured using the Dual-Glo luciferase assay system (Promega) accordingto the manufacturer's protocols.

Chromatin IP (ChIP) Assay

ChIP assays were performed as previously described (Gan, et al., GenesDev., 2011, 25(24):2619-2630). Briefly, differentiated human skeletalmyotubes were cross-linked with 1% formaldehyde for 10 minutes at roomtemperature, and glycine was added to stop the cross-linking reaction.Cells were harvested and lysed. For chromatin fragmentation, sonicationwas performed using a Bioruptor (Diagenode). Proteins wereimmunoprecipitated by using anti-MondoA (Bethyl Laboratories; catalognumber A303-195A) or IgG control (Sigma; catalog number 15006) overnightat 4° C. DNA fragments were purified using a QIAquick PCR purificationkit (Qiagen) and quantified by a LightCycler® 480 Instrument II (Roche)with the specific primers listed in Table 1.

TABLE 1 Primer sequences for ChIP-qRT-PCR. Forward (5′ to 3′)Reverse (5′ to 3′) Human promoter TXNIP CCGGGCAGCCAATGGGAGGCAGGAGGCGGAAACGTCT ChoRE (SEQ ID NO: 58) C (SEQ ID NO: 59) ARRDC4CGGAGATAACCCTGTTCC CAGGCCGTTTACTGGCTGA ChoRE GC (SEQ ID NO: 60)(SEQ ID NO: 61) Impa2 CTATCGGATGGTCAGCTT GCACTGGCTTCTCATGTTT MEF2CAA (SEQ ID NO: 62) ATC (SEQ ID NO: 63) Mouse promoter TxnipGCCTGGTAAACAAGGGCC GCTGCCGGAAACGGCTTAT ChoRE AA (SEQ ID NO: 64)A (SEQ ID NO: 65) Pklr GATCCAGGCTCTGCAGAC CAGCTAGCATCTCTCTTGC ChoREAG (SEQ ID NO: 66) CA (SEQ ID NO: 67) Myh7 TGCATACAGACTTGGTGAGAACAGTGTGAAGACTCCT Intron ATAG (SEQ ID NO: ATG (SEQ ID NO: 69) 26 68)

In Vivo Studies

All animal studies were performed in accordance with the NationalInstitute of Health guidelines for humane treatment of animals andapproved by the IACUC of the Sanford Burnham Prebys Medical DiscoveryInstitute at Lake Nona. Six week old male C57BL/6J mice were obtainedfrom Jackson Laboratory (stock number 000664) and acclimated for 1 weekprior to study. Mice were maintained on standard chow or a 60% high fatdiet (Research Diets, USA) for 8 weeks. SBI-993 was dissolved in avehicle containing 2% DMSO, 2% Tween-80, and 96% water (pH9.0). Sevenweeks after HFD feeding, mice were subcutaneously injected with SBI-993(50 mg/kg mouse body weight) once daily for 7 days. Each group contains6-10 mice and three independent experiments were conducted.

Statistics

Student's t-test, Mann Whitney test, one-way ANOVA with Bonferroni posthoc test, or two-way ANOVA with Tukey post hoc test was performed todetermine statistical significance as indicated. Non-linear regressionanalysis was used to calculate EC50 values of SBI-477 and SBI-993 in thetriglyceride accumulation assay in human skeletal myotubes (GraphPadPrism 6).

Results Identification of a Small Molecule Inhibitor of Myocyte NeutralLipid Accumulation

Hits from a cell-based high-throughput screen performed on murine AML12hepatocytes to identify molecular probes that decrease triacylglyceride(TAG) accumulation resultant from oleate loading were examined forchemical tractability and activity in human skeletal myocytes. Oneparticular compound, an N-(thiazol-2-yl)-benzamide termed SBI-477,showed potent inhibition of TAG accumulation in rat H9c2 myocytes(EC50≈100 nM, data not shown) and human skeletal myotubes (EC50≈1 μM;FIGS. 1A and 1B, data not). Inhibition of TAG accumulation by SBI-477was not due to blocking cellular fatty acid uptake or increasingintracellular lipolysis rates (FIG. 1C and data not shown). In addition,SBI-477 had no effect on the gene expression of the fatty acidtransporter, CD36 (data not shown). Increased oxidation of fatty acidscould also account for the TAG-lowering actions of SBI-477. Indeed, FAOrates were increased by SBI-477 in a dose-dependent manner concordantwith inhibition of increased triglyceride storage (FIG. 1D).Acylcarnitine species indicative of mitochondrial fatty acid oxidation(FAO) intermediates were also increased following exposure to SBI-477 inhuman myotubes consistent with increased mitochondrial FAO rates (FIG.1E). However, inhibition of FAO did not prevent the actions of SBI-477on TAG levels, even in the presence of carnitine, suggesting that thiseffect is likely downstream of the primary site of TAG-lowering action(FIGS. 1E and 1F).

To assess the effects of SBI-477 on triglyceride synthesis andremodeling, quantitative lipidomic analyses were conducted on extractsof oleate-loaded human skeletal myotubes following a 24 hour exposure toSBI-477, compared to vehicle. The effects of SBI-477 were broad,reducing all TAG species measured, with greatest effects on TAG specieswith acyl chain lengths from 16-20. 18:1 species were markedly reducedby SBI-477, consistent with oleate loading (FIGS. 2A and 2B). Levels ofTAG and DAG species were also reduced following exposure to the compound(FIG. 2A). The levels of specific lipid species implicated in insulinresistance or cellular lipotoxicity were also examined. Levels ofceramide and total sphingomyelin were not significantly altered withSBI-477 treatment in oleate-loaded myotubes. However, a significantreduction in a subset of sphingomyelin species including thosecontaining a 16:0 acyl chain was observed with SBI-477 treatment.Interestingly, however, these changes were not observed in non-oleateloaded myotubes. These results indicate that SBI-477 inhibitsincorporation of all fatty acids, including those of exogenous origin,into the neutral lipid TAG pool.

Intracellular TAG can be formed through reacylation of DAG speciesgenerated from several pathways including a de novo glycerolphosphatebiosynthetic pathway that involves dephosphorylation of phosphatidicacid (DAGPA), reacylation of monoacylglycerol (DAGMAG), and to a lesserextent, hydrolysis of phosphatidyl inositol (DAGPI). To explore theeffects of SBI-477 on specific TAG biosynthesis pathways, abioinformatics lipidomics modeling approach was used as previouslydescribed (Han, et al., Lipid Res. 2013; 54(4):1023-1032). This approachuses the individual TAG ion profile, as determined by mass spectrometry,to predict the relative contribution of each pathway to the overallcellular TAG pool. Experiments were conducted in skeletal myocytes inthe absence and presence of oleate loading. In the absence of exogenousoleic acid, SBI-477 markedly reduced contribution by the de novoglycerolphosphate biosynthetic pathway (K1) resulting in shift (10-foldincrease) to the monoacylglycerol reacylation pathway (K2) (Table 3).

TABLE 3 Triglyceride remodeling induced by SBI-477 Oleate/ PathwayBAS/Vehicle BSA/SBI-477 Oleate/Vehicle SBI-477 K1 0.96 ± 0.03 0.50 ±0.08* 0.25 ± 0.02 0.36 ± 0.03* K2 0.04 ± 0.03 0.44 ± 0.10* 0.75 ± 0.020.63 ± 0.03* K3 0.00 0.05 ± 0.03  0.00 0.01 ± 0.02 

In contrast, incubation with SBI-477 resulted in a modest decrease in K2with a shift to K1 in the oleate-loaded condition. These results,together with the lipidomic profiling data, indicate that SBI-477inhibits several cellular TAG synthesis pathways and that the mechanismfor inhibition of TAG biosynthesis by SBI-477 is distinct depending onwhether the source of fatty acid is exogenous or synthesized de novo.Notably, it was not found that SBI-477 directly inhibits the activity ofSCD-1, DGAT1/2 or MGAT1/2/3 (data not shown) which led to the discoveryof a novel inhibitory mechanism.

Stimulation of Muscle Glucose Uptake and Insulin Signaling by SBI-477

Subsequent studies sought to determine whether the IMCL-lowering effectsof the molecular probe, SBI-477, was linked to changes in myocyteglucose uptake. SBI-477 increased both basal and insulin stimulatedglucose uptake in human skeletal myotubes (approximately 84% at 10 μMSBI-477; FIG. 3A). Several compounds identified as hits from the AML12hepatocyte high throughput screen (HTS) were tested for their ability toincrease glycogen synthesis in H9c2 myotubes. Glycogen synthesis wasused as a surrogate readout for glucose uptake in these cells. As shown,only SBI-477 (MLS-0227479) increased insulin-stimulated glycogensynthesis as compared to vehicle. Several examples of hits from theAML12 hepatocyte HTS were tested for their ability to increase glycogensynthesis in H9c2 myotubes. The tested compounds were:

As shown in FIG. 3B, only SBI-477 (MLS-0227479) increasedinsulin-stimulated glycogen synthesis as compared to vehicle. Glycogensynthesis rates were enhanced by SBI-477 treatment in a dose dependentmanner (FIG. 3C). The effects on glucose uptake were independent of, butadditive with, that of insulin. Similar effects on glucose uptake wereobserved in oleate-loaded myotubes (FIG. 3D). Interestingly, SBI-477activated insulin signaling in the absence of insulin. Specifically,tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) wasincreased by SBI-477 (FIGS. 3E and 3F). Conversely, IRS-1phosphorylation at serine sites 636/639, which has been shown to conferinhibition of insulin signaling (Bouzakri, et al., Diabetes. 2003;52(6):1319-1325), was decreased by exposure to SBI-477. Phosphorylationof the downstream effector kinase, Akt, was also increased by SBI-477.The effects on insulin signaling triggered by SBI-477 were observedafter 24 hours of compound exposure but not with acute treatment (datanot shown). These results indicate that SBI-477 stimulates glucoseuptake by activating insulin signaling through a mechanism that does notrequire insulin engaged to its receptor.Signaling events downstream of insulin signaling were also assessed.Surprisingly, levels of pS6K (T389), a target of insulin—mTORC1signaling, were reduced by SBI-477 treatment (FIG. 3G). Accordingly,other signaling pathways upstream of mTORC1 were examined. Notably,SBI-477 treatment resulted in activation of AMPK, an inhibitor of mTORsignaling, consistent with the observed inhibitory phosphorylation ofS6K (FIG. 3G). It is possible, therefore, that activation of AMPKinhibits mTORC1 activity, despite activation of insulin signaling, inthe context of SBI-477 exposure.

SBI-477 Reduces Expression of TXNIP and ARRDC4, Negative Regulators ofInsulin Signaling, Via Deactivation of the Transcription Factor MondoA

To gain further insight into the downstream actions of SBI-477,transcriptional profiling were conducted in primary human skeletalmyotubes. Heat map visualization of differentially expressed transcriptsrevealed that 24 hour exposure to SBI-477 prevented many of thetranscriptomic changes resultant from oleate loading, and regulated adistinct subset of transcripts compared with that of a DGAT1 inhibitor(data not shown). Examination of the highly regulated transcriptsregulated by SBI-477, but not the DGAT inhibitor, identified thioredoxininteracting protein (TXAIP) and arrestin-domain containing 4 (ARRDC4) asmarkedly downregulated. TXNIP and ARRDC4 have been shown to function aspotent negative regulators of glucose uptake and insulin signaling(Parikh, et al. PLoS Med. 2007; 4(5):e158; Yoshihara, et al. Nat Commun.2010; 1:127). SBI-477 conferred robust, dose-dependent downregulation ofTXAIP and ARRDC4 expression in human myotubes, an effect that was alsoobserved under oleate-loaded conditions, albeit to a lesser extent(FIGS. 4A-4B). TXNIP protein levels were also reduced by SBI-477 indose-dependent manner (FIG. 4C).

Given the marked down regulation of TXNIP and ARRDC4 transcript levelsby SBI-477, effects at the transcriptional level were explored next. Forthese studies, a reporter construct containing approximately 1.5 kb ofthe human TXAIP promoter was transfected into H9c2 myocytes (Oslowski,et al., Cell Metab. 2012; 16(2):265-273). SBI-477 decreased promoteractivity in a dose dependent manner (FIG. 5A). Regulation of TXNIPexpression is known to be regulated, at least in part, by twocarbohydrate response elements (ChoRE) within its promoter region(Cha-Molstad, et al., J Biol Chem. 2009; 284(25):16898-16905).Mutational analysis confirmed that the SBI-477-mediated inhibition ofTXAIP promoter is dependent upon an intact ChoRE (FIG. 5B).

ChoREs were originally characterized as bound by heterodimers comprisedof the Carbohydrate Responsive Element-Binding Protein (ChREBP; MondoB)and Max-like protein X (MLX) transcription factors. A highly relatedprotein, MondoA is muscle-enriched and has been shown to bind to similarconsensus sites (Billin, et al. Mol Cell Biol. 2000; 20(23):8845-8854).There studies confirmed that human myotubes in culture predominantlyexpress MondoA compared to ChREBP (MondoB) using immunoblotting (datanot shown). Both ChREBP and MondoA have been shown to regulate TXAIP andARRDC4 expression (Cha-Molstad, et al., J Biol Chem. 2009;284(25):16898-16905; Stoltzman, et al., Proc Natl Acad Sci USA. 2008;105(19):6912-6917). Therefore, additional studies sought to determine ifSBI-477 affected occupation of the ChoREs located in the TXNIP andARRDC4 promoter regions. Chromatin immunoprecipitation (ChIP) studiesconfirmed occupation of MondoA on ChoREs located within both the TXNIPand ARRDC4 promoter regions but not on a control Mef2 binding site inthe IMPA2 gene promoter in human skeletal myotubes (FIG. 5C). Theoccupation by MondoA was inhibited by SBI-477 (FIG. 5C). Thus, SBI-477reduces the binding of MondoA to the TXNIP and ARRDC4 promoter regions.

The activity of ChREBP and MondoA is governed, in part, bynuclear-cytoplasmic shuttling mechanisms that are incompletelyunderstood but likely involve glucose metabolites and phosphorylation(Stoltzman, et al., Proc Natl Acad Sci USA. 2008; 105(19):6912-6917;Filhoulaud, et al., Trends Endocrinol Metab. 2013; 24(5):257-268).Accordingly, the effect of SBI-477 on intracellular localization ofMondoA was assessed. Immunolocalization studies confirmed that treatmentwith SBI-477 resulted in near complete nuclear exclusion of MondoA inhuman skeletal myoblasts (data not shown). The results of immunoblottingstudies conducted with fractionated cellular extracts further supportedthis conclusion (FIG. 5D). Taken together, these results demonstratethat downregulation of TXNIP and ARRDC4 expression downstream of SBI-477involves deactivation of MondoA via effects on nuclear localization.

MondoA Coordinately Regulates Pathways Involved in Myocyte GlucoseUptake and TAG Synthesis

siRNA-mediated knockdown (KD) studies were performed in human skeletalmyocytes to determine whether inhibition of MondoA was responsible forthe observed effects of SBI-477 on myocyte glucose uptake. FollowingMondoA (MLXJP) KD (FIG. 6G), expression of TXNIP and ARRDC4 was reducedto a similar degree as treatment with SBI-477 (FIGS. 6A and 6B). Insulinindependent glucose uptake was enhanced following MondoA KD in humanmyotubes (FIG. 6C).

MondoA KD also inhibited TAG accumulation following oleate loading (FIG.6D). Given that the lipidomic studies implicated a mechanism targetingTAG synthesis, a determination was sought as to whether theSBI-477-MondoA pathway affected the expression of genes involved in thispathway. MondoA KD reduced the expression of several genes in thelipogenesis and TAG synthesis pathways including FASN, GPAM, EVOVL6,ACACB (ACC2) and DGAT2 (FIG. 6E). A very similar, although notidentical, gene regulatory pattern was observed with SBI-477 treatment(FIG. 6F). Thus, depletion of MondoA reproduced the actions of SBI-477on both glucose and lipid metabolism.

To determine if the effects conferred by SBI-477 observed in myocytes inculture are relevant in vivo, studies were conducted in C57BL/6 micewith diet-induced obesity. For these studies, an analog of SBI-477,named SBI-993 was used, which exhibited improved potency and suitablepharmacokinetic properties for in vivo bioavailability (FIG. 6H).SBI-993 reduced TXNIP and ARRDC4 expression to a similar degree asSBI-477 in human myotubes (data not shown). Mice were fed a 60% high fatdiet (HFD) for 8 weeks resulting in significant weight gain (FIG. 6I).During the final week of HFD feeding, SBI-993 or vehicle control wasadministered as a once daily dose (50 mg/kg s.c.) for 7 days. Plasmaconcentration of SBI-993 (4.97±0.97 μM) 4 hours following the final dosewas above the cellular EC50. SBI-993 treatment resulted in a small butsignificant reduction in body weight as compared to vehicle (FIG. 6I).

The effect of SBI-993 on MondoA target gene expression was used as abiomarker to confirm its expected actions in vivo. SBI-993 treatmentreduced the expression of TAG synthesis and lipogenic genes in bothmuscle and liver (FIG. 7A). In addition, SBI-993 administration reducedTxnip and Arrdc4 expression, an effect that was especially robust inliver (FIG. 7A, bottom). ChTP analysis demonstrated that occupation ofboth ChREBP and MondoA on the Txnip and pyruvate kinase (Pklr) genepromoters was reduced in liver by SBI-993 (FIG. 7E).

Consistent with the observed actions in vitro, TAG levels weresignificantly reduced in skeletal muscle following SBI-993administration (FIG. 7B, left). Hepatic steatosis was also substantiallyameliorated with compound treatment (FIG. 7B, right and data not shown).The decreased lipid deposition in liver and skeletal muscle wasassociated with improved glucose tolerance in mice administered SBI-993(FIG. 7C). Finally, SBI-993 improved insulin signaling in both muscleand liver following an acute insulin challenge (FIGS. 7D and 7F). Thesedata demonstrate that this compound class ameliorates obesity relatedlipotoxicity, including lipid accumulation and glucose tolerance,concomitant with reduced MondoA/ChREBP signaling and improved insulinaction.

Discussion

Excessive cellular neutral lipid accumulation is a hallmark of caloricexcess and obesity. It is now well established that expansion ofintramyocellular lipid (IMCL) is strongly associated with thedevelopment of insulin resistance (Krssak, et al., Diabetologia. 1999;42(1):113-116; Pan, et al., J Clin Invest. 1995; 96(6):2802-2808; Jacob,et al., Diabetes. 1999; 48(5):1113-1119; and Coen, et al., TrendsEndocrinol Metab. 2012; 23(8):391-398). However, the intracellular lipiddepot per se is likely not directly involved in this pathologic process,as insulin sensitive conditions may also be associated with increasedIMCL such as is exhibited by endurance athletes (Goodpaster, et al., JClin Endocrinol Metab. 2001; 86(12):5755-5761). Although specific lipidspecies such as diacylglycerols (DAG) and ceramides have been shown tocontribute to muscle insulin resistance in some studies (Montell, etal., Am J Physiol Endocrinol Metab. 2001; 280(2):E229-237; Yu, et al., JBiol Chem. 2002; 277(52):50230-50236; Adams, et al. Diabetes. 2004;53(1):25-31; Bergman, et al., Diabetologia. 2012; 55(4):1140-1150;Szendroedi, et al., Proc Natl Acad Sci USA. 2014; 111(26):9597-9602.),this conclusion has not been consistently supported by all publishedwork (Skovbro, et al., Diabetologia. 2008; 51(7):1253-1260; Anastasiou,at al., Metabolism. 2009; 58(11):1636-1642; Amati, et al., Diabetes.2011; 60(10):2588-2597). In addition, the normal biological processesthat coordinately control myocyte lipid storage and glucose import arepoorly understood. Therefore, an unbiased chemical biology screen wasconducted to identify regulatory circuits that coordinately controlmyocyte lipid stores and glucose uptake. Delineation of the downstreamactions of a molecule identified in this screen, revealed that thetranscription factor, MondoA, coordinately regulates genes involved inmyocyte TAG synthesis and TXNIP and ARRDC4, known inhibitors of insulinsignaling. Thus, inhibition of MondoA by SBI-477 results in reducedcellular TAG accumulation and enhanced glucose uptake. This mechanismwas shown to be operative in vivo in skeletal muscle and liver,suggesting that this regulatory pathway is relevant to multiple celltypes.

MondoA was first described as a heterodimeric partner of the Mlxtranscription factor (Billin, et al., Mol Cell Biol. 2000;20(23):8845-8854). It has been proposed that MondoA, and its moreintensively studied hepatic-enriched relative, ChREBP (MondoB), serve asintracellular glucose and energy sensors (Filhoulaud, et al., TrendsEndocrinol Metab. 2013; 24(5):257-268; Dentin, et al., J Biol Chem.2004; 279(19):20314-20326; Peterson, et al., Mol Cell Biol. 2010;30(12):2887-2895). Increased levels of glucose-6-phosphate and othercarbohydrate intermediates have been shown to stimulate the nuclearimport of ChREBP and MondoA resulting in a feedback loop that results inthe control of glucose uptake and metabolism (Stoltzman, et al., ProcNatl Acad Sci USA. 2008; 105(19):6912-6917; Peterson, at al., Mol CellBiol. 2010; 30(12):2887-2895; Kabashima, et al., Proc Natl Acad Sci USA.2003; 100(9):5107-5112; Sakiyama, et al, J Biol Chem. 2008;283(36):24899-24908; Stoltzman, et al., J Biol Chem. 2011;286(44):38027-38034). The results described herein demonstrate thatMondoA activates expression of genes involved in myocyte TAG synthesisand de novo lipogenesis. This regulation is reminiscent of the effectsof ChREBP on hepatic lipogenesis (Dentin, et al., Diabetes. 2006;55(8):2159-2170; Iizuka, et al., Am J Physiol Endocrinol Metab. 2006;291(2):E358-364). Depletion of MondoA reduced TAG levels inoleate-loaded myocytes, an effect that mimicked the inhibitory effectsof SBI-477. This mechanism was also shown in vivo as administration ofSBI-993 resulted in a reduction in muscle TAG levels and reduced hepaticsteatosis associated with reduced expression of lipogenic and TAGsynthesis genes. MondoA suppresses myocyte glucose uptake via activationof the α-arrestin proteins TXNIP and ARRDC4, establishing a negativefeedback loop to restrict glucose entry (Stoltzman, et al., Proc NatlAcad Sci USA. 2008; 105(19):6912-6917; Kaadige, et al., Proc Natl AcadSci USA. 2009; 106(35):14878-14883).

Overexpression of either TXNIP or ARRDC4 inhibits cellular glucoseuptake (Parikh, et al., PLoS Med. 2007; 4(5):e158; Patwari, et al. JBiol Chem. 2009; 284(37):24996-25003) by repressing insulin signalingvia mechanisms that are as yet undetermined (Yoshihara, et al., NatCommun. 2010; 1:127). Interestingly, TXNIP expression is increased inhuman skeletal muscle of type 2 diabetes patients and inverselycorrelated with insulin-stimulated glucose uptake (Parikh, et al., PLoSMed. 2007; 4(5):e158;). In addition, deletion of TXNIP in the ob/obbackground improved insulin sensitivity with activation of the insulinsignaling pathway in skeletal muscle (Yoshihara, et al., Nat Commun.2010; 1:127). SBI-477 administration phenocopied the effects on TXNIPand ARRDC4 expression observed with MondoA depletion in skeletalmyocytes, resulting in enhanced cellular glucose uptake. SBI-477 exertsthis effect by markedly reducing nuclear levels of MondoA.

The MondoA-mediated mechanism described here was identified by acell-based phenotypic small molecule screen. Phenotypic screens allowfor unbiased identification of molecules that function as probes ofspecific cellular processes and mechanisms with high relevance tobiology and disease. However, a current challenge of phenotypic screensrelates to the difficulty in identifying the direct molecular target.While not being bound by theory, it is possible that SBI-477 directlytargets enzymes involved in de novo lipogenesis or TAG synthesis.Targets such as DGAT, MGAT or SCD-1 were ruled out, but the possibilitythat SBI-477 inhibits other enzymes in this or related pathways cannotbe excluded yet. However, the results are consistent with a mechanismthat imposes upstream control of MondoA signaling. SBI-477 may alsoaffect the level of an intracellular signal that controls nuclearlocalization of both MondoA and ChREBP. Indeed, previous studies haveidentified several candidate mediators for the control of ChREBP/MondoAcellular localization and activity including intermediates of glucosemetabolism, OGlcNAc modification, and phosphorylation (Peterson, at al.,Mol Cell Biol. 2010; 30(12):2887-2895, Sakiyama, et al, J Biol Chem.2008; 283(36):24899-24908; Kawaguchi; at al., Proc Natl Acad Sci USA.2001; 98(24):13710-13715; Bricambert, et al., J Clin Invest. 2010;120(12):4316-4331; Guinez, et al., Diabetes. 2011; 60(5):1399-1413).However, given the impact of SBI-477 on lipid metabolism, it is alsopossible that a lipid signal influences MondoA nuclear localization.

The results described suggest a role of MondoA signaling in muscle.MondoA may serve to maintain cellular carbon and energy homeostasis.Specifically, in states of acute fuel excess and ample energy stores,MondoA may serve to reduce fuel catabolism by triggering metaboliccheckpoint functions that redirect carbon sources by inhibiting glucoseuptake via suppression of insulin signaling, and incorporation of fattyacids into lipid storage depots (FIG. 8). MondoA has also been shown toactivate genes involved in glycogen synthesis, further supporting a rolein diversion of fuel to storage depots (Petrie, et al., Mol Cell Biol.

2013; 33(4):725-738). The known mechanisms of MondoA regulation are alsoconsistent with this notion. For example, MondoA nuclear levels areincreased by glucose metabolites, including phosphometabolites, whichcould serve as indicators of high glucose flux and ample energyphosphate stores (Petrie, et al., Mol Cell Biol. 2013; 33(4):725-738;Sloan, et al., Genes Cancer. 2010; 1(6):587-596). In addition,inhibition of oxidative phosphorylation results in deactivation ofMondoA, which would release its inhibition on glucose import and fuelcatabolic flux (Yu, et al., J Biol Chem. 2010; 285(33):25822-25830).Accordingly, MondoA may serve to limit carbon intake and fuel burningduring periods of acute fuel excess. In states of chronic caloricexcess, persistent activation of MondoA may become maladaptive (FIG. 8),contributing to a vicious cycle of cellular lipid accumulation (TAGsynthesis) and insulin resistance (TXNIP-mediated effects).

The findings demonstrate that the regulatory circuit defined in the cellstudies is operative in vivo.

Following administration of a high fat diet in mice, a structuralhomolog of SBI-477, SBI-993, reduced TAG levels in muscle, and to agreater extent in liver. The reduction of intramyocellular and hepatictriglyceride accumulation was associated with improved insulin signalingand glucose tolerance. It should be noted that the compound causedmodest weight loss. Some contribution to the metabolic effects ofSBI-993 by the modest weight loss effects cannot be excluded. Notably,the weight loss was not due to reduced food intake (data not shown)making a generalized toxic effect of the compound unlikely. In addition,the effects on tissue lipids and Txnip gene expression weredisproportionate to the modest weight loss. Interestingly, the ChIPresults demonstrated that SBI-993 reduced occupation of ChREBP inaddition to MondoA on liver target genes, suggesting that the compoundlikely affects both MondoA and ChREBP in liver. Taken together, thefindings provide a therapeutic target for insulin resistance and tissuelipotoxicity caused by chronic caloric excess.

Preparation of Compounds Using General Synthetic Schemes CompoundsPrepared Using Scheme 3

3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-((2-morpholino-2-oxoethyl)amino)benzamide(Cpd. 75)

The commercially available 3-methoxy-4-nitrobenzoic acid (1.0 eq) wasdissolved in acetonitrile (0.25 M) and treated with triethylamine (2.0eq) and HATU (1.0 eq). To this mixture was then added4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq). The resulting solution waswarmed to 65° C. and stirred until the reaction was complete. At thistime the solution was cooled to room temperature and diluted with water.The resulting precipitate was collected via filtration, washed withadditional water, and dried in vacuo to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide as a paleyellow solid (82% yield). A dry round bottom flask was charged with 10%palladium on carbon (0.05 eq) under an atmosphere of nitrogen. EtOAc(˜0.2 M) was then added, followed by3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide (1.0 eq).A hydrogen-filled balloon was then affixed to the flask and the airspacewas evacuated and back-filled with hydrogen. The mixture was vigorouslystirred until the reaction was complete, adding additional catalyst asneeded and some methanol to ensure solubility. The mixture was thenfiltered through celite, washing with methanol to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-nitrobenzamide as ayellow solid. This solid (1.0 eq) was then dissolved in acetonitrile(0.1 M) and treated with excess glyoxylic acid (˜15 eq of a 50% aqueoussolution). Sodium cyanoborohydride (5.0 eq) was then added and stirredat room temperature until the reaction was complete. The solution wasthen quenched with acetic acid, and partitioned between EtOAc and 1NHCl. The organic portion was then concentrated to a yellow solid thatwas used without further purification. The crude(2-methoxy-4-((4-(4-methoxyphenyl)thiazol-2-yl)carbamoyl) phenyl)glycine(1.0 eq) was dissolved in DMF and treated with morpholine (10.0 eq). Tothis room temperature solution was added HATU (3.0 eq) and stirring wascontinued until the reaction was complete. The solution was thenquenched with water which initiated the formation of a whiteprecipitate. This impure solid was collected via filtration and thenpurified on silica gel (hexane/EtOAc gradient 35%-100%) to give3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-((2-morpholino-2-oxoethyl)amino)benzamideas a white solid. ¹H-NMR (DMSO-d₆, 500 MHz): δ 12.33 (s, 1H), 7.88 (d,J=8.8 Hz, 2H), 7.75 (dd, J=8.3, 1.9 Hz, 1H), 7.67 (d, J=2.0 Hz, 1H),7.44 (s, 1H), 7.00 (d, J=8.9 Hz, 2H), 6.67 (d, J=8.5 Hz, 1H), 5.84 (t,J=4.6 Hz, 1H), 4.05 (d, J=4.6 Hz, 2H), 3.94 (s, 3H), 3.79 (s, 3H), 3.63(br, 2H), 3.58 (br, 2H), 3.51 (br, 4H). ¹³C-NMR (DMSO-d₆, 125 MHz): δ167.19, 164.61, 158.90, 158.87, 148.82, 145.38, 141.20, 127.36, 127.05,123.02, 118.38, 114.02, 108.83, 108.75, 106.03, 65.98, 65.90, 55.72,55.12, 44.28, 43.82, 41.87. MS [M+H]: 483.22.

N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholinoethoxy)benzamide(Cpd. 78)

A vial was charged with commercially available4-(2-morpholinoethoxy)benzoic acid (1.2 eq),4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq), and anhydrous DMF (0.15 M).To this solution was then added triethylamine (2.0 eq), then HATU (1.2eq). The mixture was then warmed to 75° C. and stirred until thereaction was complete. The solution was then cooled to room temperature,diluted with water and extracted with EtOAc. The organic portion wasthen washed with brine and dried over sodium sulfate. Concentration invacuo and purification on silica gel (CH₂Cl₂/MeOH gradient) gaveN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholinoethoxy)benzamide.¹H-NMR (MeOD-d₄, 500 MHz): δ 8.03 (d, J=8.9 Hz, 2H), 7.86 (d, J=8.7 Hz,2H), 7.25 (s, 1H), 7.11 (d, J=8.8 Hz, 2H), 6.96 (d, J=8.7 Hz, 2H), 4.25(t, J=5.3 Hz, 2H), 3.83 (s, 3H), 3.76-3.70 (m, 4H), 2.89-2.81 (m, 3H),2.62 (m, 4H). MS [M+H]: 440.31.

Compounds prepared using Scheme 4

4-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-oxobutanamide(Cpd. 81)

A microwave vial was charged with 3,4-dihydro-2H-benzo[b][1,4]oxazine(1.0 eq), succinic anhydride (1.0 eq) and acetonitrile (0.33 M) andheated at 100° C. for 30 min. To this room temperature solution was thenadded triethylamine (2.0 eq), 4-(4-methoxyphenyl)thiazol-2-amine (1.0eq), and HATU (1.2 eq). The solution was again heated to 100° C. for 45minutes. After cooling to room temperature, the solution was dilutedwith water which initiated the precipitation of a solid. This solid wascollected via filtration and then further purified on silica gel(hexane/EtOAc gradient 5%-80%) to give4-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-oxobutanamideas a white solid. ¹H-NMR (CDCl₃, 500 MHz): δ 9.81 (s, 1H), 7.75 (d,J=8.8 Hz, 2H), 7.10 (s, 1H), 6.98 (s, 1H), 6.95-6.87 (m, 4H), 4.31 (m,2H), 3.98 (br, 2H), 3.83 (s, 3H), 3.04 (br, 2H), 2.81 (m, 2H). MS [M+H]:424.28.

N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-morpholino-4-oxobutanamide (Cpd.82)

A microwave vial was charged with morpholine (1.0 eq), succinicanhydride (1.0 eq) and acetonitrile (0.4 M) and heated at 100° C. for 30min. To this room temperature solution was then added triethylamine (2.0eq), 4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq), and HATU (1.2 eq). Thesolution was again heated to 100° C. for 45 minutes. After cooling toroom temperature, the solution was diluted with water. The solid thusformed did not dissolve in water, or in EtOAc. This solid was collectedvia filtration and was shown to be cleanN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-morpholino-4-oxobutanamide. ¹H-NMR(CDCl₃, 500 MHz): δ 10.23 (s, 1H), 7.75 (d, J=8.8 Hz, 2H), 6.97 (s, 1H),6.93 (d, J=8.8 Hz, 2H), 3.84 (s, 3H), 3.69 (m Hz, 6H), 3.56-3.48 (m,2H), 2.89-2.82 (m, 2H), 2.81-2.76 (m, 2H). ¹³C-NMR (CDCl₃, 125 MHz): δ170.44, 170.35, 159.75, 157.75, 149.70, 127.56, 127.47, 114.28, 105.96,67.03, 66.63, 55.56, 45.97, 42.55, 31.60, 28.50. MS [M+H]: 376.14.

Compounds prepared using Scheme 5

A vial was charged with the arylamine (1.0 eq), triethylamine (3.0 eq),and DMF (˜0.4 M). To this solution was then added the3-methoxy-4-(methoxymethoxy)benzoic acid (1.2 eq). Finally, HATU (1.2eq) was added and the vial was warmed to 75° C. Stirring at 75° C. wascontinued until full consumption of the carboxylic acid by LCMS. Thesolution was then cooled to room temperature, diluted with EtOAc, andwashed with water and brine. Concentration in vacuo gave an oil that wasused without further purification. This oil was directly dissolved inTHE (2 mL) and treated with 1N HCl (0.5 mL). The solution was thenwarmed to 65° C. and stirred until the acetal deprotection was completeby LCMS. Upon completion, the solution was cooled to room temperature,diluted with EtOAc and sequentially washed with water and brine. Thesolution was dried over sodium sulfate, concentrated in vacuo, andpurified on silica gel (hex/EtOAc gradient) to give the clean phenol.

A vial was charged with the phenol (1.0 eq) and acetonitrile (0.3 M).Potassium carbonate (2.0 eq) was added, followed by2-chloro-1-morpholinoethan-1-one (1.0 eq). This solution was then warmedto 85° C. and stirred for 4-5 h. The solution was cooled to roomtemperature and diluted with water and twice extracted with EtOAc.Purification on silica gel (hex/EtOAc gradient 10%-100%) gave theexpected product.

A microwave vial was charged with 2-bromo-1,3-diphenylpropan-1-one (1.0eq), thiourea (1.05 eq), and ethanol (0.2 M). The mixture was heated at60° C. for 15 min and then partitioned between water anddichloromethane. The organic portion was then dried over sodium sulfateand concentrated in vacuo. Purification on silica gel gave5-benzyl-4-phenylthiazol-2-amine (69% yield). ¹H-NMR (500 MHz, CDCl₃) δ7.44-7.40 (m, 2H), 7.30 (m, 3H), 7.24-7.19 (m, 2H), 7.12 (m, 3H), 3.97(s, 2H), 3.27 (m, 2H).

A microwave vial was charged with (3-chloroprop-1-yn-1-yl)benzene (1.0eq), thiourea (1.0 eq) and acetonitrile (0.25 M). Potassium carbonate(1.0 eq) was then added and the combined mixture was heated at 110° C.for 30 min. The solution was then diluted with water and extracted withEtOAc. The organic portion was concentrated in vacuo and purified onsilica gel (hexane/EtOAc gradient 2%-65%) to give4-benzylthiazol-2-amine. ¹H-NMR (500 MHz, CDCl₃) δ 7.31 (m, 2H), 7.27(m, 2H), 7.23 (m, 1H), 6.01 (s, 1H), 5.43 (s, 2H), 3.89 (s, 2H).

A flask was charged with 2-(4-methoxyphenyl)acetyl chloride (1.0 eq) andMeCN at room temperature. Trimethylsilyl diazomethane (5.2 eq) wascarefully added dropwise to the solution. The resulting mixture wasstirred for 3 h and then concentrated in vacuo to give an orange oil.This oil was then dissolved in THE (0.5 M) and treated with HBr (33%,5.2 eq) at 0° C. After 1 h, the solution was poured into water andextracted with EtOAc. The organic portion was dried over magnesiumsulfate and concentrated in vacuo. This1-bromo-3-(4-methoxyphenyl)propan-2-one (1.0 eq) was then dissolved inEtOH and treated with thiourea (1.9 eq). The mixture was then warmed to75° C. for 1 h. The solution was partitioned between EtOAc and water,and then dried over magnesium sulfate. Concentration and purification onsilica gel (hexane/EtOAc gradient 10%-100%) gave4-(4-methoxybenzyl)thiazol-2-amine. ¹H-NMR (500 MHz, Chloroform-d) δ7.17 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 5.98 (t, J=1.1 Hz, 1H),5.08 (br, 2H), 3.80 (s, 2H), 3.79 (s, 3H).

2-(2-methoxy-4-(((4-(4-methoxyphenyl)thiazol-2-yl)amino)methyl)phenoxy)-1-morpholinoethan-1-one(Cpd. 89)

A microwave vial was charged with3-methoxy-4-(methoxymethoxy)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamide(1.0 eq) and THE (0.1 M). BH₃.THF (15 eq of a 1.0 M solution) wascarefully added and the solution was heated to 75° C. for 60 min. Thesolution was then quenched with aqueous HCl and then warmed again to 75°C. for 30 min at which time the acetal deprotection was complete. Thesolution was then diluted with EtOAc and washed with NaHCO₃ and brine.The organic portion was dried over sodium sulfate, concentrated invacuo, and purified on silica gel (hexane/EtOAc gradient 10%-40%) togive 2-methoxy-4-(((4-(4-methoxyphenyl)thiazol-2-yl)amino)methyl)phenol.This phenol (1.0 eq) was then dissolved in acetonitrile (0.1 M) andtreated with 2-chloro-1-morpholinoethan-1-one (1.0 eq) and potassiumcarbonate (2.0 eq). This mixture was heated in a microwave at 85° C. for4 h. The inorganic salts were then removed via filtration and thefiltrate was concentrated in vacuo. Purification on silica gel(hexane/acetone gradient) gave2-(2-methoxy-4-(((4-(4-methoxyphenyl)thiazol-2-yl)amino)methyl)phenoxy)-1-morpholinoethan-1-one.¹H-NMR (500 MHz, CDCl₃) δ 7.73 (d, J=8.8 Hz, 2H), 6.95 (d, J=1.7 Hz,1H), 6.93-6.88 (m, 4H), 6.57 (s, 1H), 5.70 (br, 1H), 4.73 (s, 2H), 4.43(d, J=4.2 Hz, 2H), 3.83 (s, 3H), 3.83 (s, 3H), 3.65 (m, 8H). MS [M+H]:470.25.

3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholinoethoxy)benzamide(Cpd. 90)

A microwave vial was charged with ethyl3-methoxy-4-(2-morpholino-2-oxoethoxy)benzoate (1.0 eq) and THF (0.1 M).BH₃THF (15 eq of a 1.0 M solution) was carefully added and the resultingsolution was warmed to 75° C. for 1 h. This solution was quenched withwater and concentrated in vacuo. The material was then dissolved inTHF/water (2:1) and treated with LiOH (5 eq). This mixture was thenheated at 85° C. for 1 h and concentrated in vacuo. This material wasthen treated with acetonitrile (0.1 M), triethylamine (4.0 eq) and HATU(1.0 eq). 4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq) was then added andthe combined solution was warmed to 65° C. The reaction was sluggish, soadditional HATU was added as needed to complete the reaction. Thesolution was then quenched with water and thrice extracted with EtOAc.The combined organics were dried over sodium sulfate and concentrated invacuo. Purification on silica gel (hexane/acetone gradient) gave3-methoxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholinoethoxy)benzamide.¹H-NMR (CDCl₃, 500 MHz): δ 9.59 (s, 1H), 7.75 (d, J=8.8 Hz, 2H), 7.53(d, J=2.1 Hz, 1H), 7.46 (dd, J=8.4, 2.1 Hz, 1H), 7.05 (s, 1H), 6.97-6.92(m, 3H), 4.25 (t, J=6.0 Hz, 2H), 3.93 (s, 3H), 3.85 (s, 3H), 3.76 (t,J=4.7 Hz, 4H), 2.92 (t, J=6.0 Hz, 2H), 2.66 (br, 4H). MS [M+H]: 470.23.

N-(3-methoxy-4-(2-morpholino-2-oxoethoxy)phenyl)-4-(4-methoxyphenyl)thiazole-2-carboxamide(Cpd. 91)

A microwave vial was sequentially charged with 2-methoxy-4-nitrophenol(1.0 eq), MeCN (0.2 M), 2-chloro-1-morpholinoethan-1-one (1.0 eq) andpotassium carbonate (2.0 eq). The mixture was heated at 120° C. untilthe reaction was complete, at which time the inorganic solids wereremoved via filtration and the filtrate was concentrated in vacuo togive 2-(2-methoxy-4-nitrophenoxy)-1-morpholinoethan-1-one as a yellowsolid. ¹H-NMR (500 MHz, CDCl₃) δ 7.88 (dd, J=8.9, 2.6 Hz, 1H), 7.78 (d,J=2.6 Hz, 1H), 6.99 (d, J=8.9 Hz, 1H), 4.86 (s, 2H), 3.96 (s, 3H), 3.67(t, J=4.5 Hz, 4H), 3.62 (dd, J=10.7, 4.9 Hz, 3H). A round bottom flaskwas then charged with Pd/C (10%, 0.05 eq) and EtOAc (0.2 M) under anatmosphere of nitrogen.2-(2-methoxy-4-nitrophenoxy)-1-morpholinoethan-1-one (1.0 eq) was thenadded, followed by MeOH (0.3 M) to aid in dissolution. A hydrogen-filledballoon was affixed to the flask and the mixture was vigorously stirredovernight. Upon completion, the mixture was filtered through celite andwashed with methanol. Concentration in vacuo gave2-(4-amino-2-methoxyphenoxy)-1-morpholinoethan-1-one. ¹H-NMR (500 MHz,CDCl₃) δ 6.80 (d, J=8.5 Hz, 1H), 6.30 (d, J=2.6 Hz, 1H), 6.20 (dd,J=8.4, 2.6 Hz, 1H), 4.63 (s, 2H), 3.80 (s, 3H), 3.71-3.59 (m, 8H). Amicrowave tube was charged with ethyl 2-amino-2-thioxoacetate (1.0 eq),2-bromo-1-(4-methoxyphenyl)ethan-1-one (1.0 eq), and aqueous EtOH (0.5M, 50%). The solution was then heated at 75° C. for 60 min. The solutionwas poured into water and extracted with dichloromethane. Concentrationin vacuo and purification on silica gel (hexane/EtOAc gradient 20%-50%)gave ethyl 4-(4-methoxyphenyl)thiazole-2-carboxylate. ¹H-NMR (500 MHz,CDCl₃) δ 7.90 (d, J=8.8 Hz, 2H), 7.62 (s, 1H), 6.96 (d, J=8.9 Hz, 2H),4.51 (q, J=7.1 Hz, 2H), 3.86 (s, 3H), 1.46 (t, J=7.1 Hz, 3H). Thismaterial (1.0 eq) was then dissolved in aqueous THE (0.3 M, 4:1 THF:H₂O)and treated with LiOH (4.0 eq). The mixture was stirred for 60 min, thenpoured into aqueous HCl (1 N) and extracted twice with dichloromethane.The organic portions were combined, dried over sodium sulfate, andconcentrated in vacuo to give 4-(4-methoxyphenyl)thiazole-2-carboxylicacid as a yellow solid. A vial was then charged with4-(4-methoxyphenyl)thiazole-2-carboxylic acid (1.0 eq) and2-(4-amino-2-methoxyphenoxy)-1-morpholinoethan-1-one (1.0 eq) in MeCN(0.2 M). Triethylamine (2.0 eq) and HATU (1.0 eq) were then added andthe resulting mixture was stirred at room temperature for 2 h. Thesolution was then poured into water and twice extracted withdichloromethane. Concentration and purification on silica gel(hexane/EtOAc gradient 50%-100%) gaveN-(3-methoxy-4-(2-morpholino-2-oxoethoxy)phenyl)-4-(4-methoxyphenyl)thiazole-2-carboxamide.¹H-NMR (500 MHz, Acetone-d₆) δ 10.04 (s, 1H), 8.10 (s, 1H), 7.99 (d,J=8.8 Hz, 2H), 7.68 (d, J=2.4 Hz, 1H), 7.44 (dd, J=8.7, 2.4 Hz, 1H),7.06 (d, J=8.7 Hz, 1H), 7.02 (d, J=8.8 Hz, 2H), 4.85 (s, 2H), 3.89 (s,3H), 3.85 (s, 3H), 3.61 (m, 8H). MS [M+H]: 484.18.

Compounds Prepared Using Scheme 6

A vial was charged with a phenolic carboxylic acid (1.0 eq) and MeCN(0.15 M). To this solution was then added triethylamine (3.0 eq) and a4-aryl-2-aminothiazole (1.0 eq). HATU (1.0 eq) was lastly added and thesolution was heated at 75° C. until conversion was complete. Thesolution was quenched with water which caused the precipitation of theproduct, which was used without further purification.

A microwave vial was charged with the above-prepared phenol (1.0 eq) anda 2:1 MeCN:DMF mixture. To this solution was then added2-chloro-1-morpholinoethan-1-one (1.0 eq) and potassium carbonate (2.0eq). The vial was then heated at 85° C. until alkylation was complete.The solution was then quenched with water and purified directly onreverse phase preparatory HPLC.

N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholino-2-oxoethoxy)cyclohexane-1-carboxamide(Cpd. 97)

A vial was charged with 4-oxocyclohexane-1-carboxylic acid (1.0 eq) andMeCN (0.15 M) at room temperature. To this solution was then addedtriethylamine (3.0 eq) and HATU (1.0 eq).4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq) was lastly added and thesolution was warmed to 70° C. for 4 h. The solution was then cooled toroom temperature and diluted with water which initiated theprecipitation ofN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-oxocyclohexane-1-carboxamide.¹H-NMR (500 MHz, CDCl₃) δ 7.77 (d, J=8.8 Hz, 2H), 7.06 (s, 1H), 6.97 (d,J=8.8 Hz, 2H), 3.85 (s, 3H), 2.59 (m, 1H), 2.47 (m, 2H), 2.25-2.08 (m,4H), 2.03 (m, 2H). This ketone (1.0 eq) was then dissolved indichloromethane/methanol (4:1) and treated with sodium borohydride (2.0eq) at room temperature. After 10 min the reaction was completed and thesolution was quenched with HCl (1 N). Dichloromethane was used toextract the solution two times, the combined organic portions were driedover sodium sulfate and concentrated in vacuo to give4-hydroxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)cyclohexane-1-carboxamide.This material (1.0 eq) was then dissolved in THE (0.1 M) and treatedwith potassium t-butoxide (1.1 eq) at room temperature.2-chloro-1-morpholinoethan-1-one (1.1 eq) was then added and thesolution was warmed to 60° C. until alkylation was complete. Thesolution was then poured into aqueous NH₄Cl and extracted with EtOAc.The organic portion was washed with brine and dried over sodium sulfate.Concentration in vacuo gaveN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholino-2-oxoethoxy)cyclohexane-1-carboxamideas a white solid that was washed twice with hexane and thrice with Et₂Oto give the clean product. ¹H-NMR (500 MHz, DMSO-d₆) δ 7.82 (d, J=8.3Hz, 2H), 7.44 (d, J=30.6 Hz, 1H), 6.98 (d, J=8.3 Hz, 2H), 5.27 (s, 1H),3.79 (d, J=2.0 Hz, 3H), 3.73 (s, 2H), 3.61-3.43 (m, 8H), 2.78-2.57 (m,1H), 1.93-1.77 (m, 4H), 1.56-1.40 (m, 2H), 1.31-1.03 (m, 2H). MS [M+H]:460.22

4-(ethylamino)-3-(2-hydroxyethoxy)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamide(Cpd. 99)

A vial was charged with 4-oxocyclohexane-1-carboxylic acid (1.0 eq) andMeCN (0.15 M) at room temperature. To this solution was then addedtriethylamine (3.0 eq) and HATU (1.0 eq).4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq) was lastly added and thesolution was warmed to 70° C. for 4 h. The solution was then cooled toroom temperature and diluted with water which initiated theprecipitation ofN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-oxocyclohexane-1-carboxamide.¹H-NMR (500 MHz, CDCl₃) δ 7.77 (d, J=8.8 Hz, 2H), 7.06 (s, 1H), 6.97 (d,J=8.8 Hz, 2H), 3.85 (s, 3H), 2.59 (m, 1H), 2.47 (m, 2H), 2.25-2.08 (m,4H), 2.03 (m, 2H). This ketone (1.0 eq) was then dissolved indichloromethane/methanol (4:1) and treated with sodium borohydride (2.0eq) at room temperature. After 10 min the reaction was completed and thesolution was quenched with HCl (1 N). Dichloromethane was used toextract the solution two times, the combined organic portions were driedover sodium sulfate and concentrated in vacuo to give4-hydroxy-N-(4-(4-methoxyphenyl)thiazol-2-yl)cyclohexane-1-carboxamide.This material (1.0 eq) was then dissolved in THE (0.1 M) and treatedwith potassium t-butoxide (1.1 eq) at room temperature.2-chloro-1-morpholinoethan-1-one (1.1 eq) was then added and thesolution was warmed to 60° C. until alkylation was complete. Thesolution was then poured into aqueous NH₄Cl and extracted with EtOAc.The organic portion was washed with brine and dried over sodium sulfate.Concentration in vacuo gaveN-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholino-2-oxoethoxy)cyclohexane-1-carboxamide(Cpd. 97) as a white solid that was washed twice with hexane and thricewith Et₂O to give the clean product.

A round bottom flask was charged with methyl 3-hydroxy-4-nitrobenzoate(1.0 eq) and 2-(2-bromoethoxy)tetrahydro-2H-pyran (1.5 eq) in DMF (0.4M). Potassium carbonate (2.0 eq) was then added and the mixture waswarmed to 80° C. After 5 h, the solution was diluted with EtOAc andwashed four times with water and once with brine. The solution was thendried over sodium sulfate, concentrated in vacuo, and purified on silicagel (hexane/EtOAc gradient 2%-25%) to give methyl4-nitro-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoate as a yellowoil. ¹H-NMR (500 MHz, CDCl₃) δ 7.80 (d, J=1.5 Hz, 1H), 7.78 (d, J=8.4Hz, 1H), 7.66 (dd, J=8.4, 1.5 Hz, 1H), 4.69 (t, J=3.5 Hz, 1H), 4.39-4.28(m, 2H), 4.07 (ddd, J=11.6, 5.0, 3.9 Hz, 1H), 3.93 (s, 3H), 3.83 (dtt,J=10.1, 7.2, 3.3 Hz, 2H), 3.54-3.47 (m, 1H), 1.78 (m, 1H), 1.69 (m, 1H),1.56 (m, 2H), 1.53-1.46 (m, 2H). ¹³C-NMR (126 MHz, CDCl3) δ 165.13,151.87, 142.77, 134.70, 125.14, 121.59, 116.19, 99.06, 69.66, 65.30,62.05, 52.78, 30.39, 25.36, 19.17.

A round bottom flask was charged with Pd/C (10%, 0.05 eq) under anatmosphere of nitrogen. EtOAc (0.25 M) was added, followed by methyl4-nitro-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoate (1.0 eq). Ahydrogen-filled balloon was then affixed to the flask and the airspacewas evacuated and backfilled with hydrogen three times. The mixture wasvigorously stirred at room temperature for 7 h and then filtered throughcelite, washing with methanol. Concentration in vacuo gave methyl4-amino-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoate. ¹H-NMR (500MHz, Chloroform-d) δ 7.55 (dd, J=8.2, 1.7 Hz, 1H), 7.50 (d, J=1.8 Hz,1H), 6.69 (d, J=8.2 Hz, 1H), 4.70 (t, J=3.6 Hz, 1H), 4.23 (t, J=4.8 Hz,2H), 4.07 (dt, J=11.0, 4.8 Hz, 1H), 3.94-3.79 (m, 5H), 3.58-3.48 (m,1H), 1.83 (m, 1H), 1.74 (m, 2H), 1.70-1.47 (m, 4H).

This aniline (1.0 eq) was then dissolved in THF/MeOH (2:1) and treatedwith AcOH (1.0 eq) and acetaldehyde (5.0 eq) at 0° C. NaCNBH₃ (1.5 eq)was then added and the solution was warmed to room temperature for 45min. The solution was partially concentrated in vacuo, diluted withEtOAc and sequentially washed with HCl (1 N), NaHCO₃, water, and brine.The organic portion was then dried over sodium sulfate and concentratedin vacuo to give methyl4-(ethylamino)-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoate as ayellow oil. ¹H-NMR (500 MHz, Chloroform-d) δ 7.63 (dd, J=8.4, 1.8 Hz,1H), 7.44 (d, J=1.8 Hz, 1H), 6.52 (d, J=8.3 Hz, 1H), 4.79 (br, 1H), 4.70(t, J=3.6 Hz, 1H), 4.22 (t, J=4.8 Hz, 2H), 4.07 (dt, J=11.2, 4.6 Hz,1H), 3.89 (ddd, J=11.2, 8.4, 2.9 Hz, 1H), 3.84 (s, 3H), 3.83-3.76 (m,1H), 3.57-3.49 (m, 1H), 3.22 (q, J=7.1 Hz, 2H), 1.90-1.79 (m, 1H),1.79-1.69 (m, 1H), 1.67-1.49 (m, 4H), 1.28 (t, J=7.2 Hz, 3H).

The resulting compound (1.0 eq) was then dissolved in THF/water (2:1)and treated with LiOH (4.0 eq). The resulting mixture was heated toreflux overnight. The solution was then cooled to room temperature,acidified with 10% citric acid and thrice extracted with EtOAc. Thecombined organic fractions were washed with brine and dried over sodiumsulfate. The solution was concentrated to an oil and then precipitatedfrom Et₂O/hexanes to give4-(ethylamino)-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoic acidas a white solid. ¹H-NMR (500 MHz, Chloroform-d) δ 7.73 (dd, J=8.4, 1.8Hz, 1H), 7.50 (d, J=1.7 Hz, 1H), 6.56 (d, J=8.4 Hz, 1H), 4.72 (t, J=3.6Hz, 1H), 4.24 (t, J=4.8 Hz, 2H), 4.08 (dt, J=11.2, 4.6 Hz, 1H), 3.90(ddd, J=11.2, 8.5, 2.9 Hz, 1H), 3.84 (dt, J=11.2, 4.8 Hz, 1H), 3.59-3.52(m, 1H), 3.25 (q, J=7.2 Hz, 2H), 1.85 (tdd, J=11.9, 7.1, 3.2 Hz, 1H),1.75 (ddt, J=13.1, 9.6, 3.4 Hz, 1H), 1.70-1.50 (m, 4H), 1.30 (t, J=7.2Hz, 3H).

A vial was charged with4-(ethylamino)-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzoic acid(1.0 eq) and DMF (0.1 M) at room temperature. To this solution was addedtriethylamine (2.0 eq) and 4-(4-methoxyphenyl)thiazol-2-amine (1.0 eq).Lastly, HATU (1.1 eq) was added and the solution was warmed to 85° C.The solution was poured into water and twice extracted with EtOAc. Thesolution was then dried over sodium sulfate, concentrated in vacuo andpurified on reverse phase preparatory HPLC to4-(ethylamino)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)benzamide.¹H-NMR (400 MHz, Chloroform-d) δ 7.73 (dd, J=8.6, 1.5 Hz, 2H), 7.63-7.58(m, 1H), 7.53 (d, J=1.8 Hz, 1H), 6.99-6.94 (m, 3H), 6.60 (d, J=8.4 Hz,1H), 4.71 (d, J=3.6 Hz, 1H), 4.28 (t, J=4.8 Hz, 2H), 4.10 (dt, J=9.9,4.6 Hz, 1H), 3.98-3.80 (m, 6H), 3.60-3.50 (m, 1H), 3.26 (q, J=7.1 Hz,2H), 1.91-1.71 (m, 2H), 1.69-1.50 (m, 4H), 1.31 (td, J=7.2, 1.4 Hz, 3H).

This material (1.0 eq) was dissolved in THF/1N HCl (3:1) and warmed to65° C. for 30 min. The solution was then poured into NaHCO₃ and twiceextracted with dichloromethane. The solution was concentrated andpurified on reverse phase preparatory HPLC to give4-(ethylamino)-3-(2-hydroxyethoxy)-N-(4-(4-methoxyphenyl)thiazol-2-yl)benzamide(Cpd. 99). ¹H-NMR (500 MHz, Chloroform-d) δ 7.73 (d, J=8.6 Hz, 2H), 7.55(dd, J=8.3, 1.9 Hz, 1H), 7.46 (d, J=1.9 Hz, 1H), 7.00-6.93 (m, 3H), 6.59(d, J=8.3 Hz, 1H), 4.24 (t, J=4.5 Hz, 2H), 4.05 (t, J=4.5 Hz, 2H), 3.85(s, 3H), 3.24 (q, J=7.2 Hz, 2H), 1.28 (t, J=7.2 Hz, 3H). ¹³C-NMR (126MHz, CDCl3) δ 164.41, 159.72, 159.67, 148.77, 144.92, 142.94, 127.45,126.64, 122.47, 118.05, 114.22, 110.15, 108.22, 105.96, 70.15, 61.29,55.36, 37.62, 14.52. MS [M+H]: 414.23

3-methoxy-4-(2-morpholino-2-oxoethoxy)-N-(5-(3-(trifluoromethyl)benzyl)thiazol-2-yl)benzamide(Cpd. 100)

A round bottom flask was charged with3-(3-(trifluoromethyl)phenyl)propanal (1.0 eq) and dichloromethane (0.5M) and then cooled to 0° C. To this solution was then added L-proline(0.2 eq) and N-chlorosuccinimide (1.3 eq). The resulting solution wasthen stirred at room temperature for 1.5 h. The solution was thendiluted with hexane, stirred vigorously and filtered. The filtrate wasdiluted with EtOAc and sequentially washed with NaHCO₃ and brine. Thesolution wad dried over sodium sulfate and concentrated in vacuo to givea yellow oil that was then dissolved in ethanol (0.3 M). Thiourea (1.0eq) was then added and the solution was heated to reflux until thereaction was complete. It was then cooled to room temperature,concentrated in vacuo, and partitioned between EtOAc and aqueous NaHCO₃.The solution was then concentrated and purified on silica gel to give5-(3-(trifluoromethyl)benzyl)thiazol-2-amine as a beige solid. ¹H-NMR(500 MHz, CDCl₃) δ 7.50 (d, J=7.4 Hz, 1H), 7.47 (s, 1H), 7.46-7.38 (m,2H), 6.82 (d, J=1.2 Hz, 1H), 4.02 (s, 2H). This aminothiazole (1.0 eq)was dissolved in DMF (0.1 M) and treated with triethylamine (2.0 eq) and3-methoxy-4-(methoxymethoxy)benzoic acid (1.0 eq). Lastly, HATU (1.1 eq)was added and the solution was warmed to 70° C. overnight. Uponcompletion, the solution was diluted with water and purified directly onreverse phase preparatory HPLC to give3-methoxy-4-(methoxymethoxy)-N-(5-(3-(trifluoromethyl)benzyl)thiazol-2-yl)benzamide.¹H-NMR (500 MHz, CDCl₃) δ 7.58 (d, J=1.9 Hz, 1H), 7.54-7.48 (m, 3H),7.47-7.42 (m, 2H), 7.22 (d, J=8.4 Hz, 1H), 7.03 (s, 1H), 5.30 (s, 2H),4.14 (s, 2H), 3.92 (s, 3H), 3.52 (s, 3H). The acetal was removed bydissolving the compound (1.0 eq) in THF/1N HCl (4:1) and stirring at 45°C. until the reaction was complete. The solution was then poured intoaqueous NaHCO₃ and thrice extracted with dichloromethane. The combinedorganic portions were dried over sodium sulfate and concentrated to abrown solid. ¹H-NMR (500 MHz, Chloroform-d) δ 7.54 (d, J=7.5 Hz, 2H),7.50 (d, J=7.4 Hz, 1H), 7.48-7.39 (m, 3H), 6.92 (d, J=8.1 Hz, 1H), 6.81(s, 1H), 4.09 (s, 2H), 3.86 (s, 3H). The4-hydroxy-3-methoxy-N-(5-(3-(trifluoromethyl)benzyl)thiazol-2-yl)benzamide(1.0 eq) was then transferred to a microwave tube and dissolved in MeCN(0.1 M). 2-chloro-1-morpholinoethan-1-one (1.05 eq), potassium carbonate(2.0 eq), and a catalytic amount of KI were then added and the mixturewas warmed to 85° C. for 60 min. The inorganic salts were removed viafiltration and the filtrate was purified via reverse phase preparatoryHPLC to give3-methoxy-4-(2-morpholino-2-oxoethoxy)-N-(5-(3-(trifluoromethyl)benzyl)thiazol-2-yl)benzamideas a white solid. ¹H-NMR (500 MHz, DMSO-d₆) δ 7.71-7.57 (m, 6H), 7.21(s, 1H), 6.81 (d, J=8.0 Hz, 1H), 5.20 (s, 2H), 4.14 (s, 2H), 3.82 (s,3H), 3.67 (br, 2H), 3.62 (br, 2H), 3.58 (br, 2H), 3.46 (br, 2H). MS[M+H]: 536.27

Ethyl6-(5-((4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)carbamoyl)-2-(2-morpholino-2-oxoethoxy)phenoxy)hexanoate (Cpd. 101)

A vial was charged with 3,4-dihydroxybenzoic acid (1.0 eq) and aqueousmethanol (9:1 MeOH:H₂O). To this solution was then added cesiumcarbonate (0.5 eq), gas evolution was noted. After 2.5 h, the solutionwas concentrated in vacuo, then azeotroped with toluene to further dry.The resulting solid was suspended in DMF (0.15 M) and cooled to 0° C. atwhich time benzyl bromide (1.0 eq) was added and stirred for 18 h. Thesolution was then diluted with EtOAc and sequentially washed with water,NaHCO₃, and brine. The solution was then dried over sodium sulfate andconcentrated in vacuo. Purification on silica gel (hexane/EtOAc gradient2%-50%) gave benzyl 3,4-dihydroxybenzoate (31% yield). ¹H-NMR (500 MHz,Chloroform-d) δ 7.66-7.59 (m, 2H), 7.43 (d, J=7.1 Hz, 2H), 7.39 (t,J=7.3 Hz, 2H), 7.35 (d, J=7.2 Hz, 1H), 6.91 (d, J=8.3 Hz, 1H), 5.63 (s,1H), 5.32 (s, 2H), 5.25 (s, 1H).

A vial was charged with benzyl 3,4-dihydroxybenzoate (1.0 eq), Li₂CO₃(1.0 eq) and DMF (0.4 M) and warmed to 50° C. tert-butyl 2-bromoacetate(1.0 eq) was then slowly added dropwise and stirred for 22 h. Thesolution was then cooled to room temperature, diluted with water andextracted with EtOAc. Concentration and purification on silica gel(hexane/EtOAc gradient 2%-50%) gave benzyl4-(2-(tert-butoxy)-2-oxoethoxy)-3-hydroxybenzoate. ¹H-NMR (500 MHz,Chloroform-d) δ 7.68 (d, J=2.0 Hz, 1H), 7.62-7.59 (m, 1H), 7.46-7.42 (m,2H), 7.38 (t, J=7.4 Hz, 2H), 7.36-7.30 (m, 1H), 6.88 (s, 1H), 6.86 (d,J=8.5 Hz, 1H), 5.33 (s, 2H), 4.59 (s, 2H), 1.48 (s, 9H).

Potassium carbonate (2.0 eq) was added to a solution of benzyl4-(2-(tert-butoxy)-2-oxoethoxy)-3-hydroxybenzoate (1.0 eq) and ethyl6-bromohexanoate (1.2 eq) in DMF (0.25 M). The mixture was then heatedat 80° C. for 2 h, then at 60° C. overnight. The reaction was thenpartitioned between water and EtOAc and the organic portion was driedover sodium sulfate and concentrated in vacuo. Purification on silicagel (hexane/EtOAc gradient 2%-40%) gave benzyl4-(2-(tert-butoxy)-2-oxoethoxy)-3-((6-ethoxy-6-oxohexyl)oxy)benzoate(29% yield). ¹H-NMR (400 MHz, Chloroform-d) δ 7.65 (dd, J=8.4, 2.0 Hz,1H), 7.59 (d, J=2.0 Hz, 1H), 7.46-7.41 (m, 2H), 7.41-7.33 (m, 3H), 6.77(d, J=8.5 Hz, 1H), 5.34 (s, 2H), 4.62 (s, 2H), 4.13 (q, J=7.1 Hz, 2H),4.06 (t, J=6.7 Hz, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.87 (dt, J=14.3, 6.8Hz, 2H), 1.71 (p, J=7.5 Hz, 2H), 1.51 (m, 2H), 1.47 (s, 9H), 1.25 (t,J=7.1 Hz, 3H).

A vial was charged with benzyl4-(2-(tert-butoxy)-2-oxoethoxy)-3-((6-ethoxy-6-oxohexyl)oxy)benzoate(1.0 eq) and EtOAc (0.1 M) under a nitrogen atmosphere. To this solutionwas then added Pd/C (10%, 0.1 eq) and a hydrogen-filled balloon wasaffixed to the reaction vessel. The mixture was vigorously stirred for18 h, and then filtered through celite (washing with MeOH) andconcentrated to give4-(2-(tert-butoxy)-2-oxoethoxy)-3-((6-ethoxy-6-oxohexyl)oxy)benzoicacid. ¹H-NMR (400 MHz, Chloroform-d) δ 7.67 (dd, J=8.5, 2.0 Hz, 1H),7.58 (d, J=1.9 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 4.64 (s, 2H), 4.13 (q,J=7.1 Hz, 2H), 4.08 (t, J=6.7 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H), 1.89 (dt,J=14.4, 6.8 Hz, 2H), 1.80-1.66 (m, 2H), 1.48 (s, 9H), 1.26 (t, J=7.1 Hz,3H) two protons under solvent peaks.

A reaction vial was charged with4-(2-(tert-butoxy)-2-oxoethoxy)-3-((6-ethoxy-6-oxohexyl)oxy)benzoic acid(1.0 eq) and triethylamine (2.0 eq) in MeCN. To this solution was thenadded 4-(2-chloro-4-methoxyphenyl)thiazol-2-amine (1.0 eq) followed byHATU (1.0 eq). The solution was warmed to 75° C. until the reaction wascomplete. The solution was then diluted with EtOAc and thrice washedwith water. Concentration and purification on silica gel (hexane/EtOAc5%-50% gradient) gave ethyl6-(2-(2-(tert-butoxy)-2-oxoethoxy)-5-((4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)carbamoyl)phenoxy)hexanoate.This product was then dissolved in CH₂Cl₂/TFA (4:1) and stirred at roomtemperature overnight. Concentration in vacuo gave2-(4-((4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)carbamoyl)-2-((6-ethoxy-6-oxohexyl)oxy)phenoxy)aceticacid which was used without further purification.

A flask was charged with2-(4-((4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)carbamoyl)-2-((6-ethoxy-6-oxohexyl)oxy)phenoxy)aceticacid (1.0 eq) and DMF (0.25 M) at room temperature. To this solution wasthen added triethylamine (2.0 eq) and morpholine (4.0 eq). Lastly, HATU(2.0 eq) was added and stirred for 1 h. The solution was then dilutedwith EtOAc and twice washed with water. Concentration gave a white solidwhich was purified on silica gel (hexane/EtOAc gradient 15%-100%) togive ethyl6-(5-((4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)carbamoyl)-2-(2-morpholino-2-oxoethoxy)phenoxy)hexanoate.¹H-NMR (500 MHz, DMSO-d₆) δ 12.61 (s, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.77(d, J=2.1 Hz, 1H), 7.71 (dd, J=8.5, 2.1 Hz, 1H), 7.53 (s, 1H), 7.13 (d,J=2.7 Hz, 1H), 7.03 (dd, J=8.7, 2.6 Hz, 1H), 7.00 (d, J=8.6 Hz, 1H),4.96 (s, 2H), 4.13-4.01 (m, 4H), 3.82 (s, 3H), 3.62 (br, 2H), 3.57 (br,2H), 3.51-3.43 (m, 4H), 2.69 (s, 1H), 2.33 (t, J=7.4 Hz, 2H), 1.77 (p,J=6.8 Hz, 2H), 1.62 (p, J=7.5 Hz, 2H), 1.46 (p, J=7.6, 7.2 Hz, 2H), 1.17(t, J=7.1 Hz, 3H). MS [M+H]: 646.33

N-(4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)-4-(2-morpholino-2-oxoethoxy)-3-(5-(1-(25-oxo-29-((3aR,4S,6aS)-2-oxohexahydro-]H-thieno[3,4-d]imidazol-4-yl)-3,6,9,12,15,18,21-heptaoxa-24-azanonacosyl)-1H-1,2,3-triazol-4-yl)pentanamido)benzamide(Cpd. 103)

N-(4-(2-chloro-4-methoxyphenyl)thiazol-2-yl)-3-(hept-6-ynamido)-4-(2-morpholino-2-oxoethoxy)benzamide(10 mg, 16.4 μmol) was weighed into a 4 mL vial. Biotin-PEG₇-azide wasadded as a solution in acetonitrile (0.12 mL, 100 mg/mL, 16.4 μmol)followed by Hunig's base (14.3 μL, 82 μmol) and CuI as a solution inacetonitrile (0.1 mL, 6 mg/mL, 3.3 μmol). The reaction was stirred at23° C. for 18 h. The crude product was purified by RP-HPLC and afterevaporation of the combined fractions yielded the product (13 mg, 65%)as a white foamy solid. ¹H NMR (500 MHz, Methanol-d₄) δ 8.66 (d, J=2.3Hz, 1H), 8.49 (s, 1H), 7.93 (s, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.81 (s,1H), 7.78 (dd, J=8.6, 2.3 Hz, 1H), 7.45 (s, 1H), 7.17 (d, J=8.7 Hz, 1H),7.03 (d, J=2.6 Hz, 1H), 6.93 (dd, J=8.7, 2.6 Hz, 1H), 5.01 (s, 2H), 4.52(t, J=5.0 Hz, 2H), 4.45 (dd, J=7.9, 4.8 Hz, 1H), 4.26 (dd, J=7.9, 4.4Hz, 1H), 3.84 (t, J=5.1 Hz, 2H), 3.83 (s, 3H), 3.73-3.65 (m, 4H),3.64-3.60 (m, 3H), 3.59-3.47 (m, 28H), 3.35-3.31 (m, 2H), 3.15 (ddd,J=9.0, 5.9, 4.5 Hz, 1H), 2.88 (dd, J=12.7, 5.0 Hz, 1H), 2.79-2.72 (m,2H), 2.67 (d, J=12.7 Hz, 1H), 2.57-2.5 (m, 2H), 2.18 (t, J=7.4 Hz, 2H),1.83-1.73 (m, 4H), 1.72-1.50 (m, 3H), 1.44-1.35 (m, 2H), 1.17 (t, J=7.0Hz, 1H); LRMS (ESI+ve): calculated for C₅₆H₇₉ClN₁₀O₁₅S₂[M+H]=1231.49,observed [M+H]=1231.8.

The Effect of SBI-477 Analogs on TAG Accumulation

Exemplary SBI-477 analogs disclosed above as compounds were tested fortheir effect on TAG accumulation in primary murine skeletal myocytes aspreviously described. The following compounds showed activity ontriacylglyceride (TAG) accumulation in primary murine skeletal myocytes.Activity Score (IC₅₀): A, <1 μM; B, 1 μM-10 μM; C, >10 μM

TABLE 4 Compounds based on Formula 10. Entry R1 Activity Cmpd #  1

A  7  2

B  8  3

A  9  4

A 11  5

B 12  6

C 13  7

B 14  8

C 15  9

C 16 10

B 17 11

A 18 12

B 19 13

B 20 14

B 21 15

B 10 16

A 22 17

B 23 18

C 24

TABLE 5 Activity of compounds based on Formula 12. Entry R₂ ActivityCpd. # 1 4-methoxyphenyl C 1 2 phenyl C 2 3 4-methylphenyl C 3 44-chlorophenyl B 4 5 3,4-dichlorophenyl B 5 6 4-cyanophenyl B 6

TABLE 6 Activity of compounds based on Formula 14. Entry R₂ R₃ ActivityCpd. # 2 Ph —OMe A 25 3 4-Me Ph —OMe A 30 4 4-Cl Ph —OMe A 31 5 3,4-ClPh —OMe A 37 6 4-CN Ph —OMe A 32 7 3,4-OMe Ph —OMe B 38 8 3-OMe Ph —OMeB 35 9 2,4-OMe Ph —OMe A 36 10 3,4-dioxolane Ph —OMe B 39 11 4-OCF₃ Ph—OMe A 26 12 4-NO₂ Ph —OMe A 33 13 4-pyrrolidine Ph —OMe A 34 14 4-OH Ph—OMe A 27 15 4-OEt Ph —OMe A 28 16 4-O-iPr Ph —OMe A 29 17 4-OMe Ph —H A41 18 2-Me Ph —H A 47 19 2-Cl Ph —H A 48 20 2-F Ph —H A 49 21 2-OMe Ph—H A 50 22 3-Me Ph —H A 51 23 3-Cl Ph —H A 52 24 3-F Ph —H A 53 25 2-Cl,4-OiPr Ph —H A 55 26 2-Cl, 4-OMe Ph —H A 54 27 2-F, 4-OMe Ph —H A 56 284-Et Ph —H A 44 29 4-iPr Ph —H A 45 30 4-OCHF₂ Ph —H A 40 31 4-F Ph —H A46 32 4-OCH₂CH₂OMe —H A 43

TABLE 7 Activity of compounds based on Formula 16. Entry R₁ R₂ ActivityCpd. #  1

4-OMe B 94  2

H B 95  3

3-OMe B 96  4

4-OMe C 92  5

4-OMe C 93  6

4-OMe B 90  7

4-OMe B 89  8

4-OMe C 91  9

4-OMe B 82 10

4-OMe A 81 11

4-OMe B 77 12

4-OMe B 99 13

4-OMe C 97

TABLE 8 Activity of compounds based on Formula 18. Entry R Activity Cpd.# 1

C 87 2

B 88 3

C 83 4

C 79 5

C 80 6

B 85 7

A 84 8

C 100  9

B 98

TABLE 9 Activity of compounds based on Formula 20. Entry R₁ R₂ R₃Activity Cpd. # 1 morpholine —OMe —H A 75 2 morpholine —H —H A 66 3—NHMe —H —H A 67 4 —NMe₂ —H —H A 68 5 —NHCH₂CH₂OMe —H —H C 69 6morpholine —H —Cl A 70 7 —NHMe —H —Cl A 71 8 —NMe₂ —H —Cl A 72 9—NHCH₂CH₂OMe —H —Cl A 73

TABLE 10 Activity of compounds based on Formula 22. Entry R₁ R₂ R₃Activity Cpd. # 5 —NHMe —H —Cl A 58 6 —NMe₂ —H —Cl A 60 7 —NHEt —H —Cl A59 8 —NH₂ —H —Cl B 57

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular forms and embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificforms and embodiments of the method and compositions described herein.Such equivalents are intended to be encompassed by the following claims.

1. A pharmaceutical composition comprising (i) a compound represented bythe general Formula I:A-L₁-B-L₂-D-(R₂′)_(n),   Formula I wherein A is substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl,substituted heterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,or unsubstituted alkylthio; L₁ is —C(O)NR′—, —NR′C(O)—, —C(O)O—,—OC(O)—, —O—, a bond, substituted alkyl, unsubstituted alkyl,substituted alkylene, unsubstituted alkylene, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio; L₂ is —C(O)NR′—,—NR′C(O)—, —C(O)O—, —OC(O)—, —O—, absent, a bond, substituted alkyl,unsubstituted alkyl, substituted alkylene, unsubstituted alkylene,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, substituted alkylthio, or unsubstitutedalkylthio; R′ is, for each occurrence, independently hydrogen,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstitutedC₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio; B and D areindependently substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl,unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstitutedheterocyclyl, substituted alkyl, unsubstituted alkyl, substitutedalkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstitutedalkynyl, substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, or unsubstituted alkylthio, wherein if L₂ isabsent and B and D are each independently substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, Band D are a fused ring; R₂′ is, for each occurrence, independentlyhydrogen, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstitutedC₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, or two R₂′ units fuse to form substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted C₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl,substituted heterocyclyl, or unsubstituted heterocyclyl, and wherein nis an integer between 1 and 5, inclusive; (ii) a Mondo inhibitor; or(iii) a combination of (i) and (ii); in an effective amount to reducecellular triacylglycerol (TAG) levels and/or increase cellular glucoseuptake in a subject.
 2. The composition of claim 1, wherein B and D areindependently substituted aryl, unsubstituted aryl, substitutedheteroaryl, or unsubstituted heteroaryl
 3. The composition of claim 1,wherein the compound of Formula I is represented by the general FormulaII:

wherein R₁′, R₂′, R₃′, R₄′ and R₅′ are independently hydrogen,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted C₃-C₃₀ cycloalkyl, unsubstitutedC₃-C₃₀ cycloalkyl, substituted heterocyclyl, unsubstituted heterocyclyl,substituted alkyl, unsubstituted alkyl, substituted alkenyl,unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl,substituted alkoxy, unsubstituted alkoxy, substituted amino,unsubstituted amino, substituted alkylamino, unsubstituted alkylamino,amide, substituted amide, carbonyl, substituted carbonyl, carboxyl,substituted carboxyl, substituted alkylthio, unsubstituted alkylthio,halogen (F, Cl, Br, I), hydroxyl, nitro, or cyano, or any two adjacentR₁′, R₂′, R₃′, R₄′ and R₅′ fuse to form substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedC₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substitutedheterocyclyl, or unsubstituted heterocyclyl, and X and Y are, as valencepermits, independently C, O, N, S, CR₆′R₇′, or NR₈′, wherein R₆′, R₇′,and R₈′ are independently hydrogen, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedC₃-C₃₀ cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, substituted alkylthio,or unsubstituted alkylthio.
 4. The composition of claim 3, wherein thecompound of Formula II is represented by the general Formula III:

wherein R₉′, R₁₀′, R₁₁′, R₁₂′, R₁₃′, and R₁₄′ are independently C, O, N,S, wherein the bonds between adjacent R₉′ to R₁₄′ are double or singleaccording to valency, wherein R₉′ to R₁₄′ are bound to none, one, or twohydrogens according to valency, wherein R₁₅′ is, for each occurrence,independently hydrogen, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, or cyano, and m is an integer between 1 and 12, inclusive.
 5. Thecomposition of claim 4, wherein the compound of Formula III isrepresented by the general Formula IV or general Formula V:

wherein R₁₆′, R₁₇′, R₁₈′, R₁₉′, R₂₀′, R₂₁′, R₂₂′, R₂₃′, R₂₄′, and R₂₅′are independently hydrogen, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, or cyano.
 6. The composition of claim 5, wherein the compound ofFormula IV is represented by the general Formula VIII:

wherein L₁ is —NR′C(O)—, —C(O)NR′—, or substituted amino, and R₁₈′ andR₁₉′ are independently hydrogen, substituted alkoxy, unsubstitutedalkoxy, substituted amino, or unsubstituted amino.
 7. The composition ofclaim 3, wherein the compound of Formula II is represented by thegeneral Formula IX:

wherein R₁′, R₂′ and R₃′ are independently hydrogen, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, R₁′ and R₂′, or R₂′ and R₃′ combine to formsubstituted heterocyclyl or unsubstituted heterocyclyl, and A is asubstituted alkyl, unsubstituted alkyl, substituted C₃-C₃₀ cycloalkyl,or unsubstituted C₃-C₃₀ cycloalkyl.
 8. The composition of claim 1,wherein the compound of Formula III is represented by the generalFormula VI or general Formula VII:

wherein R₁₆′, R₁₇′, R₁₈′, R₁₉′, R₂₀′, R₂₁′, R₂₂′, R₂₃′, R₂₄′, and R₂₅′are independently hydrogen, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, substituted C₃-C₃₀cycloalkyl, unsubstituted C₃-C₃₀ cycloalkyl, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, or cyano.
 9. The composition of claim 8, wherein the compound ofFormula VI is represented by the general Formula X:

wherein R₁₈′ and R₁₉′ are independently substituted alkoxy, orunsubstituted alkoxy, and R₂′ is hydrogen, substituted heterocyclyl,unsubstituted heterocyclyl, substituted alkyl, unsubstituted alkyl,substituted alkenyl, unsubstituted alkenyl, substituted alkynyl,unsubstituted alkynyl, substituted alkoxy, unsubstituted alkoxy,substituted amino, unsubstituted amino, substituted alkylamino,unsubstituted alkylamino, amide, substituted amide, carbonyl,substituted carbonyl, carboxyl, substituted carboxyl, substitutedalkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I), hydroxyl,nitro, cyano, and R₁₈′ and R₁₉′ are independently substituted alkoxy, orunsubstituted alkoxy.
 10. The composition of claim 2, wherein thecompound has the general Formula XI:

R₁′, R₂′, R₃, R₄′, and R₅′ are independently hydrogen, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, and R₁₈′ and R₁₉′ are independently substitutedalkoxy, or unsubstituted alkoxy.
 11. The composition of claim 5, whereinthe compound of Formula IV has the general Formula XII:

wherein L₂ is a bond, substituted alkylene, or unsubstituted alkylene,R₁′, R₂′, R₃, R₄′, R₅′, and R₂₅′ are independently hydrogen, substitutedheterocyclyl, unsubstituted heterocyclyl, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted alkynyl, unsubstituted alkynyl, substituted alkoxy,unsubstituted alkoxy, substituted amino, unsubstituted amino,substituted alkylamino, unsubstituted alkylamino, amide, substitutedamide, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl,substituted alkylthio, unsubstituted alkylthio, halogen (F, Cl, Br, I),hydroxyl, nitro, cyano, and R₁₈′ and R₁₉′ are independently substitutedalkoxy, or unsubstituted alkoxy.
 12. The composition of claim 1, whereinthe compound is SBI-477 or SBI-993.
 13. The composition of claim 1,wherein the Mondo inhibitor is an inhibitory antisense nucleic acid, ananti-MondoA antibody an anti-MondoB antibody, or an siRNA for MondoA, anshRNA for MondoA, an siRNA for MondoB, or shRNA for MondoB. 14.(canceled)
 15. The composition of claim 1, wherein the compositionpromotes at least one of tyrosine phosphorylation of insulin receptorsubstrate 1, nuclear translocation of MondoA, and cellular glycogensynthesis. 16.-17. (canceled)
 18. A method of reducing blood glucose ina subject, comprising administering to the subject the pharmaceuticalcomposition of claim
 1. 19. The method of claim 18, wherein the subjecthas one or more conditions selected from the group consisting ofobesity, insulin resistant obesity, heart disease, atheromatous disease,metabolic syndrome, non-alcoholic fatty liver disease including hepaticsteatosis and nonalcoholic steatohepatitis, triglyceride storagedisease, dysfunctions associated with lipid biosynthesis andtriglyceride levels, as seen for example in endurance athletes, renallipotoxicity-associated inflammation, diabetic nephropathy pancreaticbeta cell lipotoxicity-induced dysfunction, type II diabetes and insulinresistant type II diabetes.
 20. (canceled)
 21. The method of claim 18,wherein the pharmaceutical composition is an extended releaseformulation.
 22. A method reducing nuclear translocation of MondoA incancer cells in a subject comprising administering to the subject acomposition of claim 1, wherein the composition comprises a MondoAinhibitor in an effective amount to reduce nuclear translocation ofMondoA in target cancer cells.
 23. The method of claim 22, wherein thecancer is a Myc-driven cancer.
 24. The method of claim 23, wherein thecancer is selected from the group consisting of neuroblastoma, lungsquamous cell carcinoma/lung adenocarcinoma, liver hepatocellularcarcinoma, colon adenocarcinoma, acute myeloid leukemia, and breastinvasive carcinoma.
 25. A method of reducing body weight in a subjectcomprising administering to the subject the composition of claim
 1. 26.The method of claim 25, wherein the subject is obese or prediabetic.27.-39. (canceled)